Cyp11a2 is essential for oocyte development and spermatogonial stem cell differentiation in zebrafish

Endocrinology ◽  
2021 ◽  
Author(s):  
Yaqing Wang ◽  
Ding Ye ◽  
Fenghua Zhang ◽  
Ru Zhang ◽  
Junwen Zhu ◽  
...  
Keyword(s):  
Leydig Cells ◽  
Stem Cell Differentiation ◽  
High Abundance ◽  
Wild Type ◽  
Secondary Sexual Characteristics ◽  
Testicular Cells ◽  
Rate Limiting Step ◽  
Sexual Characteristics ◽  
Males And Females ◽  
Rate Limiting

Abstract Cytochrome P45011A1, encoded by Cyp11a1, converts cholesterol to pregnenolone (P5), the first and rate-limiting step in steroidogenesis. In zebrafish, cyp11a1 is maternally expressed and cyp11a2 is considered the ortholog of Cyp11a1 in mammals. A recent study has shown that depletion of cyp11a2 resulted in steroidogenic deficiencies and the mutants developed into males with feminized secondary sexual characteristics. Here, we independently generated cyp11a2 mutants in zebrafish and showed that the mutants can develop into males and females in the juvenile stage, but finally into infertile males with defective mating behavior in the adult stage. In the developing ovaries, the cyp11a2 mutation led to stage I oocyte apoptosis and final sex reversal, which could be partially rescued by treatment with P5 but not estradiol. In the developing testes, depletion of cyp11a2 resulted in dysfunction of Sertoli cells and lack of functional Leydig cells. Spermatogonial stem cells (SSCs) in the mutant testes underwent active self-renewal but no differentiation, resulting in a high abundance of SSCs in the testis, as revealed by immunofluorescence staining with Nanos2 antibody. The high abundance and differentiation competence of SSCs in the mutant testes were verified by a novel testicular cell transplantation (TCT) method developed in this study, by transplanting mutant testicular cells into germline-depleted wild-type (WT) fish. The transplanted mutant SSCs efficiently differentiated into functional spermatids in WT hosts. Overall, our study demonstrates the functional importance of cyp11a2 in early oogenesis and differentiation of SSCs.

scholarly journals A Positive Role for Rhizobitoxine in Rhizobium-legume Symbiosis

1999 ◽  
Vol 12 (12) ◽  
pp. 1082-1089 ◽  
Author(s):  
Samuel Duodu ◽  
T. V. Bhuvaneswari ◽  
Thomas J. W. Stokkermans ◽  
N. Kent Peters
Keyword(s):  
Ethylene Biosynthesis ◽  
Nodule Development ◽  
Ethylene Inhibitors ◽  
Wild Type ◽  
Positive Role ◽  
Rate Limiting Step ◽  
Mutant Strains ◽  
Legume Symbiosis ◽  
Exogenous Ethylene ◽  
Rate Limiting

Although Bradyrhizobium elkanii is a mutualistic symbiont of legumes, it synthesizes a phytotoxin, rhizobitoxine, that causes chlorosis on a variety of legume hosts, giving a pathogenic character to these interactions. No positive role for rhizobitoxine has been previously demonstrated. Interestingly, rhizobitoxine inhibits the rate-limiting step for ethylene biosynthesis, a plant hormone known to inhibit or down-regulate nodule development. We hypothesized that rhizobitoxine plays a positive role in nodule development through its inhibition of ethylene biosynthesis. To test this hypothesis, host plants of B. elkanii were screened for a differential nodulation response to the wild-type and rhizobitoxine mutant strains. In Vigna radiata (mungbean), the rhizobitoxine mutant strains induced many aborted nodules arrested at all stages of pre-emergent and post-emergent development and formed significantly fewer mature nodules than the wild type. Experiments revealed that nodulation of mungbean plants is sensitive to exogenous ethylene, and that the ethylene inhibitors aminoethoxyvinylglycine and Co2+ were able to partially restore a wild-type nodulation pattern to the rhizobitoxine mutants. This is the first demonstration of a nodulation phenotype of the rhizobitoxine mutants and suggests that rhizobitoxine plays a positive and necessary role in Rhizobium-legume symbiosis through its inhibition of ethylene biosynthesis.

Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70

2010 ◽  
Vol 432 (3) ◽  
pp. 495-506 ◽  
Author(s):  
Lionel Vercheval ◽  
Cédric Bauvois ◽  
Alexandre di Paolo ◽  
Franck Borel ◽  
Jean-Luc Ferrer ◽  
...  
Keyword(s):  
Active Site ◽  
Chloride Ions ◽  
Side Chain ◽  
Wild Type ◽  
Post Translational Modification ◽  
Rate Limiting Step ◽  
Class D ◽  
The Individual ◽  
Rate Limiting

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl–enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.

scholarly journals Mutations in VPS16 and MRT1Stabilize mRNAs by Activating an Inhibitor of the Decapping Enzyme

1999 ◽  
Vol 19 (11) ◽  
pp. 7568-7576 ◽  
Author(s):  
Shuang Zhang ◽  
Carol J. Williams ◽  
Kevin Hagan ◽  
Stuart W. Peltz
Keyword(s):  
Rna Stability ◽  
Wild Type ◽  
Mrna Decapping ◽  
Hsp70 Family ◽  
Rate Limiting Step ◽  
Nonsense Codon ◽  
Decapping Enzyme ◽  
Rate Limiting

ABSTRACT Decapping is a rate-limiting step in the decay of many yeast mRNAs; the activity of the decapping enzyme therefore plays a significant role in determining RNA stability. Using an in vitro decapping assay, we have identified a factor, Vps16p, that regulates the activity of the yeast decapping enzyme, Dcp1p. Mutations in the VPS16 gene result in a reduction of decapping activity in vitro and in the stabilization of both wild-type and nonsense-codon-containing mRNAs in vivo. The mrt1-3 allele, previously shown to affect the turnover of wild-type mRNAs, results in a similar in vitro phenotype. Extracts from both vps16 and mrt1 mutant strains inhibit the activity of purified Flag-Dcp1p. We have identified a 70-kDa protein which copurifies with Flag-Dcp1p as the abundant Hsp70 family member Ssa1p/2p. Intriguingly, the interaction with Ssa1p/2p is enhanced in strains with mutations in vps16 ormrt1. We propose that Hsp70s may be involved in the regulation of mRNA decapping.

scholarly journals A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 309-320 ◽  
Author(s):  
K Kawakami ◽  
S Pande ◽  
B Faiola ◽  
D P Moore ◽  
J D Boeke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Processing ◽  
Ribonuclease H ◽  
Wild Type ◽  
Ribosomal Frameshifting ◽  
Translational Frameshifting ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Ty1 Retrotransposition ◽  
Rate Limiting

Abstract Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C. Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for optimum frameshifting. The present work shows that the absence of tRNA-Arg(CCU) affects Ty1 transposition, translational frameshifting, and accumulation of mature TYB1 proteins. Transposition of genetically tagged Ty1 elements decreases at least 50-fold and translational frameshifting increases 3-17-fold in cells lacking tRNA-Arg(CCU). Accumulation of Ty1-integrase and Ty1-reverse transcriptase/ribonuclease H is defective in an hsx1 mutant. The defect in Ty1 transposition is complemented by the wild-type HSX1 gene or a mutant tRNA-Arg(UCU) gene containing a C for T substitution in the first position of the anticodon. Overexpression of TYA1 stimulates Ty1 transposition 50-fold above wild-type levels when the level of transposition is compared in isogenic hsx1 and HSX1 strains. Thus, the HSX1 gene determines the ratio of the TYA1 to TYA1-TYB1 precursors required for protein processing or stability, and keeps expression of TYB1 a rate-limiting step in the retrotransposition cycle.

scholarly journals Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin

2006 ◽  
Vol 399 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Zhen Shi ◽  
Gloria C. Ferreira
Keyword(s):  
Protoporphyrin Ix ◽  
Kinetic Mechanism ◽  
Wild Type ◽  
Rate Limiting Step ◽  
Transition State Analogue ◽  
Mammalian Cell Cultures ◽  
Kinetic Mechanisms ◽  
Specificity Constant ◽  
Order Of Magnitude ◽  
Rate Limiting

Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248–257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (Kiapp values of 1 μM and 2.3 μM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/Km, protoporhyrin IX) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.

scholarly journals Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1

2009 ◽  
Vol 297 (1) ◽  
pp. C66-C74 ◽  
Author(s):  
Ersin Selcuk Unal ◽  
Rongbao Zhao ◽  
I. David Goldman
Keyword(s):  
Proton Translocation ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Wild Type ◽  
Folate Transport ◽  
Rate Limiting Step ◽  
Proximal Small Intestine ◽  
Transport Cycle ◽  
Rate Limiting

The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [3H]methotrexate (MTX) influx Vmax; the MTX influx Ktwas identical to that of wild-type (WT)-PCFT (1.5 μM). Consistent with the intrinsic functionality of E185A-PCFT, [3H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (∼38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.

Revision of the oribatid mite genus Austronothrus Hammer (Acari:Oribatida):sexual dimorphism and a re-evaluation of the phylogenetic relationships of the family Crotoniidae

2009 ◽  
Vol 23 (2) ◽  
pp. 87 ◽  
Author(s):  
Matthew J. Colloff ◽  
Stephen L. Cameron
Keyword(s):  
New Zealand ◽  
Cladistic Analysis ◽  
Oribatid Mite ◽  
Morphological Characters ◽  
Separate Species ◽  
Secondary Sexual Characteristics ◽  
Sister Clade ◽  
The Family ◽  
Sexual Characteristics ◽  
Males And Females

The Gondwanan relict oribatid mite family Crotoniidae contains the genera Austronothrus Hammer, 1966, Crotonia Thorell, 1876 and Holonothrus Wallwork, 1963. This family is of considerable interest biogeographically and also because the members of the family may have re-evolved sexuality from thelytokous parthenogenetic ancestors. Crotonia and Holonothrus are speciose and widely distributed whereas Austronothrus is obscure, hitherto monospecific and known only from New Zealand. We revise Austronothrus and compare it with its better-known congeners. Two new species of Austronothrus are described from New Zealand, namely A. clarki, sp. nov., and A. flagellatus, sp. nov. The only previously known species, Austronothrus curviseta Hammer, 1966, is redescribed and the genus is redefined. The presence of male specimens of all species confirmed Austronothrus as a sexual genus, like its congeners. Secondary sexual characteristics within Austronothrus and Crotonia include significant differences in body size and proportion, as well as the lengths and shapes of various setae, predominantly those of the notogastral region. Because these characters are frequently used by taxonomists to separate and define species within the Crotoniidae, it is important that the sex of adult specimens be determined to avoid describing conspecific males and females as separate species. A phylogenetic analysis based on morphological characters shows the crotoniid genera nested within the Camisiidae, with Camisia von Heyden, 1826 basal to the crotoniid clade of Austronothrus, Holonothrus and Crotonia, and with a clade of Platynothrus Berlese, 1913, Heminothrus Berlese, 1913 and Paracamisia Olszanowski & Norton, 2002 (Camisiidae) forming the sister clade of Camisia + Crotoniidae. Thus, Camisiidae is paraphyletic with respect to Crotoniidae. The family Crotoniidae is revised in light of the cladistic analysis, and relegated to subfamily rank. Three subfamilies are defined within the Camisiidae : Crotoniinae (containing Crotonia, Austronothrus and Holonothrus), Camisiinae (containing Camisia) and Heminothridae, subfam. nov. (containing Heminothrus, Platynothrus and Neonothrus Forsslund, 1955). Nothrus maximus Trägårdh, 1901 belongs to the genus Platynothrus and is recombined.

scholarly journals Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles

IUCrJ ◽  
2014 ◽  
Vol 1 (2) ◽  
pp. 129-135 ◽  
Author(s):  
Mayank Aggarwal ◽  
Bhargav Kondeti ◽  
Chingkuang Tu ◽  
C. Mark Maupin ◽  
David N. Silverman ◽  
...  
Keyword(s):  
Binding Sites ◽  
Carbonic Anhydrases ◽  
Wild Type ◽  
Rate Limiting Step ◽  
Multiple Binding Sites ◽  
Zinc Metalloenzymes ◽  
Ping Pong ◽  
Multiple Binding ◽  
Proton Shuttling ◽  
Rate Limiting

Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO2and HCO3−, respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH−/H2O) in/out of the active siteviaHis64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type) of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1-methylimidazole, 2-methylimidazole and 4-methylimidazole) have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the `in' and `out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations) the activity of H64A CA II.

scholarly journals Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice

2006 ◽  
Vol 27 (3) ◽  
pp. 1146-1157 ◽  
Author(s):  
Chantal Kress ◽  
Carole Gautier-Courteille ◽  
H. Beverley Osborne ◽  
Charles Babinet ◽  
Luc Paillard
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Leydig Cells ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Cell Number ◽  
Pcr Analysis ◽  
Wild Type ◽  
Mammalian Development ◽  
Males And Females

ABSTRACT CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1 − / − mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1 − / − males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1 − / − males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.

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