Targeted deletion of the ATP binding domain of left-right dynein confirms its role in specifying development of left-right asymmetries

Development ◽  
1999 ◽  
Vol 126 (23) ◽  
pp. 5495-5504 ◽  
Author(s):  
D.M. Supp ◽  
M. Brueckner ◽  
M.R. Kuehn ◽  
D.P. Witte ◽  
L.A. Lowe ◽  
...  
Keyword(s):  
Motor Function ◽  
Single Amino Acid ◽  
Atp Binding ◽  
Amino Acid Difference ◽  
Sequence Classification ◽  
Ciliated Cells ◽  
Targeted Deletion ◽  
Targeted Mutation ◽  
And Function ◽  
Shed Light

Vertebrates develop distinct asymmetries along the left-right axis, which are consistently aligned with the anteroposterior and dorsoventral axes. The mechanisms that direct this handed development of left-right asymmetries have been elusive, but recent studies of mutations that affect left-right development have shed light on the molecules involved. One molecule implicated in left-right specification is left-right dynein (LRD), a microtubule-based motor protein. In the LRD protein of the inversus viscerum (iv) mouse, there is a single amino acid difference at a conserved position, and the lrd gene is one of many genes deleted in the legless (lgl) mutation. Both iv and lgl mice display randomized left-right development. Here we extend the analysis of the lrd gene at the levels of sequence, expression and function. The complete coding sequence of the lrd gene confirms its classification as an axonemal, or ciliary, dynein. Expression of lrd in the node at embryonic day 7.5 is shown to be symmetric. At embryonic day 8.0, however, a striking asymmetric expression pattern is observed in all three germ layers of the developing headfold, suggesting roles in both the establishment and maintenance of left-right asymmetries. At later times, expression of lrd is also observed in the developing floorplate, gut and limbs. These results suggest function for LRD protein in both ciliated and non-ciliated cells, despite its sequence classification as axonemal. In addition, a targeted mutation of lrd was generated that deletes the part of the protein required for ATP binding, and hence motor function. The resulting left-right phenotype, randomization of laterality, is identical to that of iv and lgl mutants. Gross defects in ciliary structure were not observed in lrd/lrd mutants. Strikingly, however, the monocilia on mutant embryonic node cells were immotile. These results prove the identity of the iv and lrd genes. Further, they argue that LRD motor function, and resulting nodal monocilia movement, are required for normal left-right development.

scholarly journals Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region

1997 ◽  
Vol 323 (3) ◽  
pp. 777-783 ◽  
Author(s):  
Éva BAKOS ◽  
Izabella KLEIN ◽  
Ervin WELKER ◽  
Katalin SZABÓ ◽  
Marianna MÜLLER ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Amino Acid ◽  
Atpase Activity ◽  
Single Amino Acid ◽  
Membrane Insertion ◽  
Atp Binding ◽  
Mdr1 Expression ◽  
And Function ◽  
Atp Binding Cassette

A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531–543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1–drug interactions directly.

scholarly journals Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2005
Author(s):  
Irene Vorontsova ◽  
James E. Hall ◽  
Thomas F. Schilling ◽  
Noriaki Nagai ◽  
Yosuke Nakazawa
Keyword(s):  
Cell Adhesion ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Adhesion Assay ◽  
Loss Of Function ◽  
Adhesive Properties ◽  
The Difference ◽  
In Vitro Adhesion ◽  
Cell To Cell Adhesion

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

scholarly journals MFng Is Dispensable for Mouse Pancreas Development and Function

2009 ◽  
Vol 29 (8) ◽  
pp. 2129-2138 ◽  
Author(s):  
Per Svensson ◽  
Ingela Bergqvist ◽  
Stefan Norlin ◽  
Helena Edlund
Keyword(s):  
Cell Differentiation ◽  
Notch Signaling ◽  
Pancreas Development ◽  
Loss Of Function ◽  
Pancreatic Cell ◽  
Targeted Deletion ◽  
Mouse Pancreas ◽  
Pancreatic Progenitor ◽  
Pancreatic Progenitor Cells ◽  
And Function

ABSTRACT Notch signaling regulates pancreatic cell differentiation, and mutations of various Notch signaling components result in perturbed pancreas development. Members of the Fringe family of β1,3-N-acetylglucosaminyltransferases, Manic Fringe (MFng), Lunatic Fringe (LFng), and Radical Fringe (RFng), modulate Notch signaling, and MFng has been suggested to regulate pancreatic endocrine cell differentiation. We have characterized the expression of the three mouse Fringe genes in the developing mouse pancreas between embryonic days 9 and 14 and show that the expression of MFng colocalized with the proendocrine transcription factor Ngn3. In contrast, the expression of LFng colocalized with the exocrine marker Ptf1a, whereas RFng was not expressed. Moreover, we show that expression of MFng is lost in Ngn3 mutant mice, providing evidence that MFng is genetically downstream of Ngn3. Gain- and loss-of-function analyses of MFng by the generation of mice that overexpress MFng in early pancreatic progenitor cells and mice with a targeted deletion of MFng provide, however, evidence that MFng is dispensable for pancreas development and function, since no pancreatic defects in these mice were observed.

scholarly journals Emerging strategies to target RAS signaling in human cancer therapy

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Kun Chen ◽  
Yalei Zhang ◽  
Ling Qian ◽  
Peng Wang
Keyword(s):  
Human Cancer ◽  
Ras Signaling ◽  
Targeting Therapy ◽  
Ras Protein ◽  
Patients With Cancer ◽  
Mutational Status ◽  
Poor Differentiation ◽  
And Function ◽  
Nsclc Patients ◽  
Shed Light

AbstractRAS mutations (HRAS, NRAS, and KRAS) are among the most common oncogenes, and around 19% of patients with cancer harbor RAS mutations. Cells harboring RAS mutations tend to undergo malignant transformation and exhibit malignant phenotypes. The mutational status of RAS correlates with the clinicopathological features of patients, such as mucinous type and poor differentiation, as well as response to anti-EGFR therapies in certain types of human cancers. Although RAS protein had been considered as a potential target for tumors with RAS mutations, it was once referred to as a undruggable target due to the consecutive failure in the discovery of RAS protein inhibitors. However, recent studies on the structure, signaling, and function of RAS have shed light on the development of RAS-targeting drugs, especially with the approval of Lumakras (sotorasib, AMG510) in treatment of KRASG12C-mutant NSCLC patients. Therefore, here we fully review RAS mutations in human cancer and especially focus on emerging strategies that have been recently developed for RAS-targeting therapy.

P53/miR-34a/SIRT1 Positive Feedback Loop regulates cell proliferation and promotes cell apoptosis in the termination stage of liver regeneration.

2021 ◽  
Author(s):  
Junhua Gong ◽  
Minghua Cong ◽  
Hao Wu ◽  
Menghao Wang ◽  
He Bai ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Liver Regeneration ◽  
Positive Feedback ◽  
Cell Apoptosis ◽  
Feedback Loop ◽  
Late Phase ◽  
Liver Weight ◽  
Positive Feedback Loop ◽  
And Function ◽  
Shed Light

Abstract Background The capacity of the liver to restore its architecture and function assures good prognoses of patients who suffer serious hepatic injury or cancer resection. In our study, we found that the P53/miR-34a/SIRT1 positive feedback loop has a remarkable negative regulatory effect, which is related to the termination of liver regeneration. Here, we described how P53/miR-34a/SIRT1 positive feedback loop controls liver regeneration and its possible relationship with liver cancer.Method We performed partial hepatectomy (PH) in mice transfected with adenovirus (Ade) overexpressing P53 and adenovirus-associated virus (AAV) knock-downing miR-34a. LR was analyzed by liver weight/body weight, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and cell proliferation, and the related cellular signals were investigated. Bile acid (BA) levels during LR were analyzed by metabolomics of bile acids. Results We found that the P53/miR-34a/SIRT1 positive feedback loop was activated in the late phase of LR. Overexpression of P53 terminated LR early and enhanced P53/miR-34a/SIRT1 positive feedback loop expression and its proapoptotic effect. Mice from the Ade-P53 group showed smaller livers and higher levels of serum ALT and AST than control mice. While knock-down of miR-34a abolished P53/miR-34a/SIRT1 positive feedback loop during LR. Mice from anti-miR-34a group showed larger livers and lower levels of PCNA-positive cells than control mice. T-β-MCA increased gradually during LR and peaked at 7 days after PH. T-β-MCA inhibited cell proliferation and promoted cell apoptosis via facilitating the P53/miR-34a/SIRT1 positive feedback loop during LR by suppressing FXR/SHP. Conclusion The P53/miR-34a/SIRT1 positive feedback loop plays an important role in the termination of LR. Our findings shed light on the molecular and metabolic mechanisms of LR termination and provide a potential therapeutic alternative for treating P53-wild-type HCC patients.

scholarly journals Role of the ATP-Binding Cassette TransporterAbcg2in the Phenotype and Function of Cardiac Side Population Cells

2008 ◽  
Vol 103 (8) ◽  
pp. 825-835 ◽  
Author(s):  
Otmar Pfister ◽  
Angelos Oikonomopoulos ◽  
Konstantina-Ioanna Sereti ◽  
Regina L. Sohn ◽  
Darragh Cullen ◽  
...  
Keyword(s):  
Side Population ◽  
Atp Binding ◽  
Side Population Cells ◽  
And Function ◽  
Atp Binding Cassette

scholarly journals Reverse evolution leads to genotypic incompatibility despite functional and active site convergence

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Miriam Kaltenbach ◽  
Colin J Jackson ◽  
Eleanor C Campbell ◽  
Florian Hollfelder ◽  
Nobuhiko Tokuriki
Keyword(s):  
Active Site ◽  
Adaptive Landscape ◽  
Sequence Structure ◽  
Activity Increase ◽  
Enzyme Active Site ◽  
Fundamental Relationship ◽  
And Function ◽  
Alternative Set ◽  
Shed Light ◽  
New Mutations

Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks.

scholarly journals Unexpected cryptic species among streptophyte algae most distant to land plants

2021 ◽  
Vol 288 (1963) ◽  
Author(s):  
Iker Irisarri ◽  
Tatyana Darienko ◽  
Thomas Pröschold ◽  
Janine M. R. Fürst-Jansen ◽  
Mahwash Jamy ◽  
...  
Keyword(s):  
Land Plants ◽  
Comparative Genomic ◽  
Future Studies ◽  
Form And Function ◽  
Genomic Studies ◽  
Plant Form ◽  
Phylogenomic Analyses ◽  
And Function ◽  
Shed Light ◽  
Green Lineage

Streptophytes are one of the major groups of the green lineage (Chloroplastida or Viridiplantae). During one billion years of evolution, streptophytes have radiated into an astounding diversity of uni- and multicellular green algae as well as land plants. Most divergent from land plants is a clade formed by Mesostigmatophyceae, Spirotaenia spp. and Chlorokybophyceae. All three lineages are species-poor and the Chlorokybophyceae consist of a single described species, Chlorokybus atmophyticus. In this study, we used phylogenomic analyses to shed light into the diversity within Chlorokybus using a sampling of isolates across its known distribution. We uncovered a consistent deep genetic structure within the Chlorokybus isolates, which prompted us to formally extend the Chlorokybophyceae by describing four new species. Gene expression differences among Chlorokybus species suggest certain constitutive variability that might influence their response to environmental factors. Failure to account for this diversity can hamper comparative genomic studies aiming to understand the evolution of stress response across streptophytes. Our data highlight that future studies on the evolution of plant form and function can tap into an unknown diversity at key deep branches of the streptophytes.

scholarly journals Immune Checkpoints Expression in Chronic Lung Allograft Rejection

2021 ◽  
Vol 12 ◽  
Author(s):  
Ilaria Righi ◽  
Valentina Vaira ◽  
Letizia Corinna Morlacchi ◽  
Giorgio Alberto Croci ◽  
Valeria Rossetti ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Bronchiolitis Obliterans Syndrome ◽  
Immune Checkpoints ◽  
Hla Antibodies ◽  
Restrictive Allograft Syndrome ◽  
And Function ◽  
Shed Light

Chronic lung allograft dysfunction (CLAD) is the main cause of poor survival and low quality of life of lung transplanted patients. Several studies have addressed the role of dendritic cells, macrophages, T cells, donor specific as well as anti-HLA antibodies, and interleukins in CLAD, but the expression and function of immune checkpoint molecules has not yet been analyzed, especially in the two CLAD subtypes: BOS (bronchiolitis obliterans syndrome) and RAS (restrictive allograft syndrome). To shed light on this topic, we conducted an observational study on eight consecutive grafts explanted from patients who received lung re-transplantation for CLAD. The expression of a panel of immune molecules (PD1/CD279, PDL1/CD274, CTLA4/CD152, CD4, CD8, hFoxp3, TIGIT, TOX, B-Cell-Specific Activator Protein) was analyzed by immunohistochemistry in these grafts and in six control lungs. Results showed that RAS compared to BOS grafts were characterized by 1) the inversion of the CD4/CD8 ratio; 2) a higher percentage of T lymphocytes expressing the PD-1, PD-L1, and CTLA4 checkpoint molecules; and 3) a significant reduction of exhausted PD-1-expressing T lymphocytes (PD-1pos/TOXpos) and of exhausted Treg (PD-1pos/FOXP3pos) T lymphocytes. Results herein, although being based on a limited number of cases, suggest a role for checkpoint molecules in the development of graft rejection and offer a possible immunological explanation for the worst prognosis of RAS. Our data, which will need to be validated in ampler cohorts of patients, raise the possibility that the evaluation of immune checkpoints during follow-up offers a prognostic advantage in monitoring the onset of rejection, and suggest that the use of compounds that modulate the function of checkpoint molecules could be evaluated in the management of chronic rejection in LTx patients.

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