scholarly journals Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues

2021 ◽  
Vol 22 (5) ◽  
pp. 2661
Author(s):  
Małgorzata Prokopowicz ◽  
Adam Jarmuła ◽  
Yannick Casamayou-Boucau ◽  
Fiona Gordon ◽  
Alan Ryder ◽  
...  
Keyword(s):  
Electrostatic Potential ◽  
Thymidylate Synthase ◽  
Wild Type Mouse ◽  
Inhibitory Mechanism ◽  
Wild Type ◽  
Theoretical Investigations ◽  
Ligand Interactions ◽  
Poisson Boltzmann

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.

Determination of the migratory capacity of embryonic cortical cells lacking the transcription factor Pax-6

Development ◽  
1997 ◽  
Vol 124 (24) ◽  
pp. 5087-5096 ◽  
Author(s):  
D. Caric ◽  
D. Gooday ◽  
R.E. Hill ◽  
S.K. McConnell ◽  
D.J. Price
Keyword(s):  
Transcription Factor ◽  
Ventricular Zone ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Cortical Cells ◽  
Developmental Potential ◽  
Neuronal Precursors ◽  
Cortical Precursors

The cerebral cortex forms by the orderly migration and subsequent differentiation of neuronal precursors generated in the proliferative ventricular zone. We studied the role of the transcription factor Pax-6, which is expressed in the ventricular zone, in cortical development. Embryos homozygous for a mutation of Pax-6 (Small eye; Sey) had abnormalities suggesting defective migration of late-born cortical precursors. When late-born Sey/Sey precursors were transplanted into wild-type embryonic rat cortex, they showed similar integrative, migrational and differentiative abilities to those of transplanted wild-type mouse precursors. These results suggest that postmitotic cortical cells do not need Pax-6 to acquire the capacity to migrate and differentiate, but that Pax-6 generates a cortical environment that permits later-born precursors to express their full developmental potential.

scholarly journals The Na+/H+ exchanger isoform 3 is required for active paracellular and transcellular Ca2+ transport across murine cecum

2013 ◽  
Vol 305 (4) ◽  
pp. G303-G313 ◽  
Author(s):  
Juraj Rievaj ◽  
Wanling Pan ◽  
Emmanuelle Cordat ◽  
R. Todd Alexander
Keyword(s):  
Water Movement ◽  
Water Flux ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Rt Pcr ◽  
Ussing Chambers ◽  
Voltage Clamps ◽  
Paracellular Absorption ◽  
Mouse Intestine

Intestinal calcium (Ca2+) absorption occurs via paracellular and transcellular pathways. Although the transcellular route has been extensively studied, mechanisms mediating paracellular absorption are largely unexplored. Unlike passive diffusion, secondarily active paracellular Ca2+ uptake occurs against an electrochemical gradient with water flux providing the driving force. Water movement is dictated by concentration differences that are largely determined by Na+ fluxes. Consequently, we hypothesized that Na+ absorption mediates Ca2+ flux. NHE3 is central to intestinal Na+ absorption. NHE3 knockout mice (NHE3−/−) display impaired intestinal Na+, water, and Ca2+ absorption. However, the mechanism mediating this latter abnormality is not clear. To investigate this, we used Ussing chambers to measure net Ca2+ absorption across different segments of wild-type mouse intestine. The cecum was the only segment with net Ca2+ absorption. Quantitative RT-PCR measurements revealed cecal expression of all genes implicated in intestinal Ca2+ absorption, including NHE3. We therefore employed this segment for further studies. Inhibition of NHE3 with 100 μM 5-( N-ethyl- N-isopropyl) amiloride decreased luminal-to-serosal and increased serosal-to-luminal Ca2+ flux. NHE3−/− mice had a >60% decrease in luminal-to-serosal Ca2+ flux. Ussing chambers experiments under altered voltage clamps (−25, 0, +25 mV) showed decreased transcellular and secondarily active paracellular Ca2+ absorption in NHE3−/− mice relative to wild-type animals. Consistent with this, cecal Trpv6 expression was diminished in NHE3−/− mice. Together these results implicate NHE3 in intestinal Ca2+ absorption and support the theory that this is, at least partially, due to the role of NHE3 in Na+ and water absorption.

scholarly journals Novel Role of MCP-Induced Protein 1 in Ischemic Brain Damage in Animals of Transient Focal Ischemia

2018 ◽  
Author(s):  
Zhuqing Jin ◽  
Jian Liang ◽  
Jiaqi Li ◽  
Pappachan E. Kolattukudy
Keyword(s):  
Cerebral Ischemia ◽  
Matrix Metalloproteinases ◽  
Tight Junction ◽  
Western Blot ◽  
Blood Brain Barrier ◽  
Wild Type Mouse ◽  
Brain Barrier ◽  
Wild Type ◽  
Blood Brain

Focal cerebral ischemia can lead to blood-brain barrier (BBB) breakdown, which is implicated in neuroinflammation and elevation of matrix metalloproteinases (MMPs). The role of the anti-inflammatory protein, monocyte chemotactic protein–induced protein 1 (MCPIP1) plays in the injury of BBB in stroke has not yet been reported. This study was conducted to identify and characterize the role MCPIP1 plays in BBB breakdown. Transient middle cerebral artery occlusion (MCAO) is induced in both wild-type and Mcpip1-/- mice for 2 hours of occlusion periods followed by reperfusion for 24 or 48 hours. BBB permeability was measured by FITC-dextran extravasation, MMP-9/3 expression was analyzed by western blot, and claudin-5 and zonula occludens-1 (ZO-1) were analyzed by immunohistochemistry and western blot. After MCAO in wild type mouse is induced, there is significantly increase in MCPIP1 mRNA and protein levels. Absence of MCPIP1 leaded to significant increase in FITC-dextran leakage in peri-infarct brain, significant upregulation of MMP-9, MMP-3 and reduced levels of tight junction components, claudin-5 and ZO-1 in the brain after MCAO. Our data demonstrate that absence of MCPIP1 exacerbates ischemia-induced blood-brain barrier disruption by enhancing the expression of matrix metalloproteinases and degradation of tight junction proteins. Overall data indicate that MCPIP1 is important protective role against BBB disruption in cerebral ischemia.

scholarly journals Diagnostic and Treatment Approaches Involving Transthyretin in Amyloidogenic Diseases

2019 ◽  
Vol 20 (12) ◽  
pp. 2982 ◽  
Author(s):  
Gil Yong Park ◽  
Angelo Jamerlan ◽  
Kyu Hwan Shim ◽  
Seong Soo A. An
Keyword(s):  
Cerebrospinal Fluid ◽  
Genetic Mutations ◽  
Hormone Binding ◽  
Wild Type ◽  
Autonomic Motor ◽  
Hormone Binding Protein ◽  
The Brain ◽  
Tetrameric Form

Transthyretin (TTR) is a thyroid hormone-binding protein which transports thyroxine from the bloodstream to the brain. The structural stability of TTR in tetrameric form is crucial for maintaining its original functions in blood or cerebrospinal fluid (CSF). The altered structure of TTR due to genetic mutations or its deposits due to aggregation could cause several deadly diseases such as cardiomyopathy and neuropathy in autonomic, motor, and sensory systems. The early diagnoses for hereditary amyloid TTR with cardiomyopathy (ATTR-CM) and wild-type amyloid TTR (ATTRwt) amyloidosis, which result from amyloid TTR (ATTR) deposition, are difficult to distinguish due to the close similarities of symptoms. Thus, many researchers investigated the role of ATTR as a biomarker, especially its potential for differential diagnosis due to its varying pathogenic involvement in hereditary ATTR-CM and ATTRwt amyloidosis. As a result, the detection of ATTR became valuable in the diagnosis and determination of the best course of treatment for ATTR amyloidoses. Assessing the extent of ATTR deposition and genetic analysis could help in determining disease progression, and thus survival rate could be improved following the determination of the appropriate course of treatment for the patient. Here, the perspectives of ATTR in various diseases were presented.

scholarly journals Complement Receptor 1 Expression on Mouse Erythrocytes Mediates Clearance of Streptococcus pneumoniae by Immune Adherence

2010 ◽  
Vol 78 (7) ◽  
pp. 3129-3135 ◽  
Author(s):  
Jie Li ◽  
Jennifer P. Wang ◽  
Ionita Ghiran ◽  
Anna Cerny ◽  
Alexander J. Szalai ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Direct Evidence ◽  
Wild Type Mouse ◽  
Complement Receptor ◽  
Blood Clearance ◽  
Wild Type ◽  
Immune Adherence ◽  
Complement Receptor 1

ABSTRACT Complement-containing immune complexes can be presented to phagocytes by human erythrocytes bearing complement receptor 1 (CR1). Although this has long been assumed to be a mechanism by which humans are able to protect themselves from “extracellular” bacteria such as pneumococci, there is little direct evidence. In these studies we have investigated this question by comparing results for erythrocytes from transgenic mice expressing human CR1 on their erythrocytes to the results for wild-type mouse erythrocytes that do not express CR1. We demonstrate that human CR1 expression on murine erythrocytes allows immune adherence to beads opsonized with either mouse or human serum as a source of complement. The role of CR1 in immune adherence was supported by studies showing that it was blocked by the addition of antibody to human CR1. Furthermore, human CR1 expression enhances the immune adherence of opsonized pneumococci to erythrocytes in vitro, and the pneumococci attached to erythrocytes via CR1 can be transferred in vitro to live macrophages. Even more importantly, we observed that if complement-opsonized pneumococci are injected intravenously with CR1+ mouse erythrocytes into wild-type mice (after a short in vitro incubation), they are cleared faster than opsonized pneumococci similarly injected with wild-type mouse erythrocytes. Finally, we have shown that the intravenous (i.v.) injection of pneumococci into CR1+ mice also results in more rapid blood clearance than in wild-type mice. These data support that immune adherence via CR1 on erythrocytes likely plays an important role in the clearance of opsonized bacteria from human blood.

BION-2: Predicting Positions of Non-Specifically Bound ions on Protein Surface by a Gaussian-based Treatment of Electrostatic Environment

2020 ◽  
Author(s):  
Mihiri Shashikala ◽  
Arghya Chakravorty ◽  
Shailesh Pandey ◽  
Emil Alexov
Keyword(s):  
Thermal Fluctuation ◽  
Computational Method ◽  
Ion Concentration ◽  
Biological Processes ◽  
Poisson Boltzmann ◽  
Concentration Changes ◽  
Energy Of Interaction ◽  
Poisson Boltzmann Equation

Abstract Background: Ions play significant roles in biological processes - they may specifically bind to a protein site or bind non-specifically on its surface. Though, the role of specifically bound ions range from actively providing structural compactness via coordination of charge-charge interactions to numerous enzymatic activities, non-specifically surface-bound ions are also crucial to maintaining a protein’s stability, responding to pH and ion concentration changes and contributing to other biological processes. However, experimental determination of positions of non-specifically bound ions is not trivial since they may have low residential time and experience significant thermal fluctuation of their positions. Results: Here we report a new release of a computational method, the BION-2 method, that predicts positions of non-specifically surface-bound ions. The BION-2 utilizes the Gaussian-based treatment of ions within the framework of the modified Poisson-Boltzmann equation, that does not require a sharp boundary between the protein and water phase. Thus, the predictions are done by the balance of the energy of interaction between the protein charges and the corresponding ions, and the de-solvation penalty of the ions as they approach the protein. Conclusions: The BION-2 is tested against experimentally determined ion’s positions, with both X-ray and NMR determined positions, and it is demonstrated that it outperforms the old BION and molecular dynamics tools. The BION-2 is available as a web server as well.

scholarly journals A Zinc Ion Controls Assembly and Stability of the Major Capsid Protein of Rotavirus

2003 ◽  
Vol 77 (6) ◽  
pp. 3595-3601 ◽  
Author(s):  
Inge Erk ◽  
Jean-Claude Huet ◽  
Mariela Duarte ◽  
Stéphane Duquerroy ◽  
Felix Rey ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Chelating Agent ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
High Concentration ◽  
Mutant Proteins

ABSTRACT The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH <7.0, about the pK of histidine, wild-type and mutated VP6 proteins display similar properties, giving rise to identical tubular and spherical assemblies. However, at pH >7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH2-terminal sequencing of the proteolytic fragments showed that they all contained the β-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the α-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.

scholarly journals Cardiac-Specific PID1 Overexpression Enhances Pressure Overload-Induced Cardiac Hypertrophy in Mice

2015 ◽  
Vol 35 (5) ◽  
pp. 1975-1985 ◽  
Author(s):  
Yaoqiu Liu ◽  
Yahui Shen ◽  
Jingai Zhu ◽  
Ming Liu ◽  
Xing Li ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Mapk Pathway ◽  
Pressure Overload ◽  
Wild Type Mouse ◽  
Heart Tissue ◽  
Wild Type ◽  
Heart Transplants ◽  
Over Expression

Background/Aims: PID1 was originally described as an insulin sensitivity relevance protein, which is also highly expressed in heart tissue. However, its function in the heart is still to be elucidated. Thus this study aimed to investigate the role of PID1 in the heart in response to hypertrophic stimuli. Methods: Samples of human failing hearts from the left ventricles of dilated cardiomyopathy (DCM) patients undergoing heart transplants were collected. Transgenic mice with cardiomyocyte-specific overexpression of PID1 were generated, and cardiac hypertrophy was induced by transverse aortic constriction (TAC). The extent of cardiac hypertrophy was evaluated by echocardiography as well as pathological and molecular analyses of heart samples. Results: A significant increase in PID1 expression was observed in failing human hearts and TAC-treated wild-type mouse hearts. When compared with TAC-treated wild-type mouse hearts, PID1-TG mouse showed a significant exacerbation of cardiac hypertrophy, fibrosis, and dysfunction. Further analysis of the signaling pathway in vivo suggested that these adverse effects of PID1 were associated with the inhibition of AKT, and activation of MAPK pathway. Conclusion: Under pathological conditions, over-expression of PID1 promotes cardiac hypertrophy by regulating the Akt and MAPK pathway.

scholarly journals Tamm-Horsfall protein-deficient thick ascending limbs promote injury to neighboring S3 segments in an MIP-2-dependent mechanism

2011 ◽  
Vol 300 (4) ◽  
pp. F999-F1007 ◽  
Author(s):  
Tarek M. El-Achkar ◽  
Ruth McCracken ◽  
Michael Rauchman ◽  
Monique R. Heitmeier ◽  
Ziyad Al-Aly ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
Wild Type Mouse ◽  
Neutrophil Infiltration ◽  
Apical Surface ◽  
Dependent Manner ◽  
Tubular Injury ◽  
Wild Type ◽  
Direct Role

Tamm-Horsfall protein (THP) is a glycoprotein expressed exclusively in thick ascending limbs (TAL) of the kidney. We recently described a novel protective role of THP against acute kidney injury (AKI) via downregulation of inflammation in the outer medulla. Our current study investigates the mechanistic relationships among the status of THP, inflammation, and tubular injury. Using an ischemia-reperfusion model in wild-type and THP−/− mice, we demonstrate that it is the S3 proximal segments but not the THP-deficient TAL that are the main targets of tubular injury during AKI. The injured S3 segments that are surrounded by neutrophils in THP−/− mice have marked overexpression of neutrophil chemoattractant MIP-2 compared with wild-type counterparts. Neutralizing macrophage inflammatory protein-2 (MIP-2) antibody rescues S3 segments from injury, decreases neutrophil infiltration, and improves kidney function in THP−/− mice. Furthermore, using immunofluorescence volumetric imaging of wild-type mouse kidneys, we show that ischemia alters the intracellular translocation of THP in the TAL cells by partially shifting it from its default apical surface domain to the basolateral domain, the latter being contiguous to the basolateral surface of S3 segments. Concomitant with this is the upregulation, in the basolateral surface of S3 segments, of the scavenger receptor SRB-1, a putative receptor for THP. We conclude that TAL affects the susceptibility of S3 segments to injury at least in part by regulating MIP-2 expression in a THP-dependent manner. Our findings raise the interesting possibility of a direct role of basolaterally released THP on regulating inflammation in S3 segments.

scholarly journals BION-2: Predicting Positions of Non-Specifically Bound Ions on Protein Surface by a Gaussian-Based Treatment of Electrostatics

2020 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
H. B. Mihiri Shashikala ◽  
Arghya Chakravorty ◽  
Shailesh Kumar Panday ◽  
Emil Alexov
Keyword(s):  
Thermal Fluctuation ◽  
Computational Method ◽  
Ion Concentration ◽  
Biological Processes ◽  
Poisson Boltzmann ◽  
Concentration Changes ◽  
Energy Of Interaction ◽  
Poisson Boltzmann Equation

Ions play significant roles in biological processes—they may specifically bind to a protein site or bind non-specifically on its surface. Although the role of specifically bound ions ranges from actively providing structural compactness via coordination of charge–charge interactions to numerous enzymatic activities, non-specifically surface-bound ions are also crucial to maintaining a protein’s stability, responding to pH and ion concentration changes, and contributing to other biological processes. However, the experimental determination of the positions of non-specifically bound ions is not trivial, since they may have a low residential time and experience significant thermal fluctuation of their positions. Here, we report a new release of a computational method, the BION-2 method, that predicts the positions of non-specifically surface-bound ions. The BION-2 utilizes the Gaussian-based treatment of ions within the framework of the modified Poisson–Boltzmann equation, which does not require a sharp boundary between the protein and water phase. Thus, the predictions are done by the balance of the energy of interaction between the protein charges and the corresponding ions and the de-solvation penalty of the ions as they approach the protein. The BION-2 is tested against experimentally determined ion’s positions and it is demonstrated that it outperforms the old BION and other available tools.

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