Effects of surfaces and macromolecular crowding on bimolecular reaction rates

2019 ◽  
Author(s):  
Steven S. Andrews
Keyword(s):  
Reaction Rates ◽  
Bimolecular Reaction ◽  
Complex Environments ◽  
Perfect Agreement ◽  
Confined Spaces ◽  
Separate Model ◽  
Planar Membranes ◽  
Reaction Rate Equation

AbstractBiological cells are complex environments that are densely packed with macromolecules and subdivided by membranes, both of which affect the rates of chemical reactions. It is well known that crowding reduces the volume available to reactants, which increases reaction rates, and also inhibits reactant diffusion, which decreases reaction rates. This work investigates these effects quantitatively using analytical theory and particle-based simulations. A reaction rate equation based on only these two processes turned out to be inconsistent with simulation results. However, accounting for diffusion inhibition by the surfaces of nearby obstacles, which affects access to reactants, led to perfect agreement for reactions near impermeable planar membranes and improved agreement for reactions in crowded spaces. A separate model that quantified reactant occlusion by crowders, and extensions to a thermodynamic “cavity” model proposed by Berezhkovskii and Szabo (J. Phys. Chem. B 120:5998, 2016), were comparably successful. These results help elucidate reaction dynamics in confined spaces and improve prediction of in vivo reaction rates from in vitro ones.

scholarly journals mTORC1 controls phase-separation and the biophysical properties of the cytoplasm by tuning crowding

2017 ◽  
Author(s):  
M. Delarue ◽  
G.P. Brittingham ◽  
S. Pfeffer ◽  
I.V. Surovtsev ◽  
S. Ping-lay ◽  
...  
Keyword(s):  
Phase Separation ◽  
Fluorescent Protein ◽  
Effective Diffusion ◽  
Reaction Rates ◽  
Biophysical Properties ◽  
Cell Interior ◽  
Profound Impact ◽  
Mtorc1 Pathway

Summary (Abstract)Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed Genetically Encoded Multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein. GEMs self-assemble into bright, stable fluorescent particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can tune the effective diffusion coefficient of macromolecules ≥15 nm in diameter more than 2-fold without any discernable effect on the motion of molecules ≥5 nm. These mTORCI-dependent changes in crowding and rheology affect phase-separation both in vitro and in vivo. Together, these results establish a role for mTORCI in controlling both the biophysical properties of the cytoplasm and the phase-separation of biopolymers.

IL-9 Production by Regulatory T Cells Recruits Mast Cells That Are Essential for Regulatory T Cell-Induced Immune-Suppression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2782-2782
Author(s):  
Anna Maria Wolf ◽  
Dominik Wolf ◽  
Andrew McKenzie ◽  
Marcus Maurer ◽  
Alexander R Rosenkranz ◽  
...  
Keyword(s):  
T Cells ◽  
Mast Cells ◽  
Regulatory T Cells ◽  
Adoptive Transfer ◽  
Conflicts Of Interest ◽  
Cell Populations ◽  
Perfect Agreement ◽  
Immunosuppressive Cell

Abstract Abstract 2782 Tipping the balance between effector and regulatory cell populations is of critical importance in the pathogenesis of various autoimmune disorders. Both, mast cells (MC) and regulatory T cells (Treg) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MC and Treg have been shown to play a protective role. We recently provided evidence that adoptive transfer of wild-type (wt) Treg into wt recipients almost completely prevents development of NTS. We here show that Treg transfer induces a profound increase of MC in the kidney draining lymph nodes (LN). In contrast, transfer of wt Treg into animals deficient in MC, which are characterized by an exaggerated susceptibility to NTS, do not prevent acute renal inflammation. Blocking the pleiotropic cytokine IL-9, which is known to be critically involved in MC recruitment and proliferation, by means of an antagonizing monoclonal antibody in animals receiving wt Treg abrogated protection from NTS. Moreover, we provide clear evidence that Treg-derived IL-9 is critical for MC recruitment as mediators of their full immune-suppressive potential, as adoptive transfer of IL-9 deficient Treg failed to protect from NTS. In line with our hypothesis, absence of Treg-derived IL-9 does not induce MC accumulation into kidney-draining LN, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Treg, as shown by in vitro testing of their functional capacities. Finally, we observed a significantly decreased expression of the MC chemoattractant Cxcl-1 in the LN of mice receiving IL-9 deficient Treg as compared to mice receiving wt Treg or control CD4+CD25− T cells, which might at least in part explain the deficient MC recruitment under these conditions. In summary, our data provide the first evidence that the immunosuppressive effects of adoptively transferred Treg depend on IL-9-mediated recruitment of MC to the kidney draining LN in NTS. This data is in perfect agreement with our previous report showing that CCR7-mediated LN occupancy of Treg is a prerequisite for their immune-suppressive potential and furthers adds a piece of information to the functional understanding of the in vivo anti-inflammatory effects of Treg. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Accurate and sensitive quantification of protein-DNA binding affinity

2018 ◽  
Vol 115 (16) ◽  
pp. E3692-E3701 ◽  
Author(s):  
Chaitanya Rastogi ◽  
H. Tomas Rube ◽  
Judith F. Kribelbauer ◽  
Justin Crocker ◽  
Ryan E. Loker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Biophysical Model ◽  
Regulatory Sequences ◽  
Binding Modes ◽  
Perfect Agreement ◽  
Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Orally Active Inhibitors of the Imatinib Resistant Bcr-Abl Mutant T315I.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2180-2180 ◽  
Author(s):  
William C. Shakespeare ◽  
Frank Wang ◽  
Qihong Xu ◽  
Xiaotian Zhu ◽  
Narayana Narasimham ◽  
...  
Keyword(s):  
Oral Administration ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Tumor Regression ◽  
Point Mutations ◽  
Drug Binding ◽  
Separate Model ◽  
Daily Oral Administration

Abstract Resistance to the Bcr-Abl kinase inhibitor imatinib in patients with chronic myeloid leukemia (CML) is associated with emergence of Bcr-Abl point mutations that preclude effective drug binding. Although most mutants are effectively inhibited with the second generation inhibitors dasatinib and nilotinib, neither compound inhibits the T315I mutant which represents ~ 25% of all clinically observed mutants. Through our program of structure-guided design, we have identified a series of compounds that inhibit the T315I mutant of Bcr-Abl both in vitro and in vivo. AP24534, a representative member of this new series, inhibited the kinase activity of both the wild-type enzyme and the T315I point mutant with IC50s of 3 and 31 nM respectively, and inhibited the proliferation of their respective BaF3-derived cell lines with IC50s of 2 and 14 nM. Additionally, AP24534 inhibited the proliferation of BaF3 cells expressing the clinically relevant mutants Y253F, E255K, H396P, or M351T with IC50s of 2, 7, 1, and 1 nM respectively. Inhibition of cellular proliferation directly correlated with decreased cellular phosphorylation of Bcr-Abl. Daily oral administration of AP24534 to mice bearing subcutaneous xenografts of Bcr-Abl-T315I-expressing BaF3 cells elicited dose-dependent tumor shrinkage, with complete tumor regression observed at the highest doses. In a separate model, daily oral administration of AP24534 significantly prolonged the survival of mice injected intravenously with these cells, at doses ranging from 5–30 mg/kg. These data indicate that this class of inhibitors has the potential to address CML refractory to current targeted agents.

Biotin synthase mechanism: an overview

2005 ◽  
Vol 33 (4) ◽  
pp. 820-823 ◽  
Author(s):  
M. Lotierzo ◽  
B. Tse Sum Bui ◽  
D. Florentin ◽  
F. Escalettes ◽  
A. Marquet
Keyword(s):  
Active Form ◽  
Reaction Rates ◽  
Similar Reaction ◽  
Sulphur Atom ◽  
Alternative Substrate ◽  
Radical Sam ◽  
Biotin Synthase ◽  
In Vitro System

Biotin synthase, a member of the ‘radical SAM’ (S-adenosylmethionine) family, converts DTB (dethiobiotin) into biotin. The active form of the Escherichia coli enzyme contains two (Fe-S) centres, a (4Fe-4S) and a (2Fe-2S). The (4Fe-4S)2+/+ mediates the electron transfer required for the reductive cleavage of SAM into methionine and a DOA• (deoxyadenosyl radical). Two DOA•, i.e. two SAM molecules, are consumed to activate the positions 6 and 9 of DTB. A direct transfer of isotope from the labelled substrate into DOAH (deoxyadenosine) has been observed with 2H, although not quantitatively, but not with tritium. The source of the sulphur introduced to form biotin is still under debate. We have shown that the (2Fe-2S)2+ cluster can be reconstituted in the apoenzyme with S2− and Fe2+. When S2− was replaced by [34S2−], [35S2−] or Se2−, biotin containing mostly the sulphur isotopes or selenium was obtained. This leads us to favour the hypothesis that the (2Fe-2S) centre is the sulphur donor, which may explain the absence of turnover of the enzyme. DTBSH (9-mercaptodethiobiotin), which already contains the sulphur atom of biotin, was shown to be an alternative substrate of biotin synthase both in vivo and with a crude extract. When this compound was tested with a well-defined in vitro system, the same turnover of one and similar reaction rates were observed for DTB and DTBSH. We postulate that the same intermediate is formed from both substrates.

scholarly journals Analysis of the size dependence of macromolecular crowding shows that smaller is better

2015 ◽  
Vol 112 (26) ◽  
pp. 7990-7995 ◽  
Author(s):  
Kim A. Sharp
Keyword(s):  
Small Molecules ◽  
Size Dependence ◽  
Reaction Rates ◽  
Medium Size ◽  
In Vitro Experiments ◽  
High Concentration ◽  
Concentration Effects ◽  
Large Molecules

The aqueous milieu inside cells contains as much as 30–40% dissolved protein and RNA by volume. This large concentration of macromolecules is expected to cause significant deviations from solution ideality. In vivo biochemical reaction rates and equilibria might differ significantly from those measured in the majority of in vitro experiments that are performed at much lower macromolecule concentrations. Consequently crowding, a nonspecific phenomenon believed to arise from the large excluded volume of these macromolecules, has been studied extensively by experimental and theoretical methods. However, the relevant theory has not been applied consistently. When the steric effects of macromolecular crowders and small molecules like water and ions are treated on an equal footing, the effect of the macromolecules is opposite to that commonly believed. Large molecules are less effective at crowding than water and ions. There is also a surprisingly weak dependence on crowder size. Molecules of medium size, ∼5 Å radius, have the same effect as much larger macromolecules like proteins and RNA. These results require a reassessment of observed high-concentration effects and of strategies to mimic in vivo conditions with in vitro experiments.

scholarly journals Tetradecanoic Acids With Anti-Virulence Properties Increase the Pathogenicity of Pseudomonas aeruginosa in a Murine Cutaneous Infection Model

2021 ◽  
Vol 10 ◽  
Author(s):  
Martha María Juárez-Rodríguez ◽  
Humberto Cortes-López ◽  
Rodolfo García-Contreras ◽  
Bertha González-Pedrajo ◽  
Miguel Díaz-Guerrero ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Cell Communication ◽  
Infection Model ◽  
Cutaneous Infection ◽  
Complex Environments ◽  
Promising Alternative ◽  
Virulence Target

Blocking virulence is a promising alternative to counteract Pseudomonas aeruginosa infections. In this regard, the phenomenon of cell-cell communication by quorum sensing (QS) is an important anti-virulence target. In this field, fatty acids (FA) have gained notoriety for their role as autoinducers, as well as anti-virulence molecules in vitro, like some saturated FA (SAFA). In this study, we analyzed the anti-virulence activity of SAFA with 12 to18 carbon atoms and compared their effect with the putative autoinducer cis-2-decenoic acid (CDA). The effect of SAFA on six QS-regulated virulence factors and on the secretion of the exoenzyme ExoU was evaluated. In addition, a murine cutaneous infection model was used to determine their influence on the establishment and damage caused by P. aeruginosa PA14. Dodecanoic (lauric, C12:0) and tetradecanoic (myristic, C14:0) acids (SAFA C12-14) reduced the production of pyocyanin by 35–58% at 40 and 1,000 µM, while CDA inhibited it 62% at a 3.1 µM concentration. Moreover, the SAFA C12-14 reduced swarming by 90% without affecting biofilm formation. In contrast, CDA reduced the biofilm by 57% at 3 µM but did not affect swarming. Furthermore, lauric and myristic acids abolished ExoU secretion at 100 and 50 µM respectively, while CDA reduced it by ≈ 92% at 100 µM. Remarkably, the coadministration of myristic acid (200 and 1,000 µM) with P. aeruginosa PA14 induced greater damage and reduced survival of the animals up to 50%, whereas CDA to 500 µM reduced the damage without affecting the viability of the PA14 strain. Hence, our results show that SAFA C12-14 and CDA have a role in regulation of P. aeruginosa virulence, although their inhibition/activation molecular mechanisms are different in complex environments such as in vivo systems.

scholarly journals Destruction of liver haem by norethindrone. Conversion into green pigments

1981 ◽  
Vol 196 (2) ◽  
pp. 575-583 ◽  
Author(s):  
Ian N. H. White
Keyword(s):  
Metabolic Activation ◽  
Fold Increase ◽  
Reaction Rates ◽  
Green Pigment ◽  
Factors Affecting ◽  
Short Period ◽  
Metabolic Transformation ◽  
Green Pigments

1. Factors affecting the norethindrone-mediated conversion of hepatic haem into green pigments have been studied in the rat. Concentrations of haem and green pigments were estimated spectrophotometrically after esterification and separation by silica gel high-pressure liquid chromatography (h.p.l.c.). 2. Accumulation of green pigments in the liver was dependent on the dose of steroid and the time after dosing, maximum values being reached after 4–8h. Phenobarbitone pretreatment of rats resulted in an 8-fold increase in the concentration of green pigments at these times. 3. In microsomal systems in vitro, the formation of green pigments in the presence of NADPH and norethindrone was also dependent on the concentration of steroid and incubation times. Reaction rates very rapidly became non-linear with time, consistent with the self-catalysed destruction of the form(s) of cytochrome P-450 responsible for the metabolic activation of norethindrone. Microsomal mixtures incubated for a short period of time (1min) with norethindrone gave only one green-pigment peak after h.p.l.c. Longer incubation times gave four or five additional green pigments. Results suggested that multiple green pigments may arise by metabolic transformation of a single precursor. 4. When liver haem was prelabelled with 14C by using 5-amino[4-14C]laevulinic acid, subsequent dosing with norethindrone in vivo gave rise to three major 14C-labelled-green-pigment peaks on h.p.l.c. None of these components had the same retention times as the green pigments produced by microsomal fractions in vitro. 5. When liver haem was prelabelled with 59Fe by using 59FeCl3, norethindrone administration resulted in the detection of 59Fe-labelled green pigments if subsequent esterification was carried out under neutral conditions with trimethyloxonium tetrafluoroborate, but not when carried out under acidic conditions with methanol/H2SO4. These results suggested that green pigments normally contain chelated iron and that metal-free green pigments are not produced by the liver.

scholarly journals Anthelmintic resistance of Haemonchus contortus from sheep flocks in Brazil: concordance of in vivo and in vitro (RESISTA-Test©) methods

2021 ◽  
Vol 30 (2) ◽  
Author(s):  
Yousmel Alemán Gainza ◽  
Isabella Barbosa dos Santos ◽  
Amanda Figueiredo ◽  
Leonardo Aparecido Lima dos Santos ◽  
Sérgio Novita Esteves ◽  
...  
Keyword(s):  
Larval Development ◽  
Haemonchus Contortus ◽  
Anthelmintic Resistance ◽  
Perfect Agreement ◽  
Fecal Egg Count ◽  
And Control ◽  
Total Agreement ◽  
Larval Development Test

Abstract This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

scholarly journals The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

2013 ◽  
Vol 33 (2) ◽  
Author(s):  
Jeremy D. Weaver ◽  
Huanchen Wang ◽  
Stephen B. Shears
Keyword(s):  
Reaction Mechanism ◽  
Atpase Activity ◽  
Adenine Nucleotide ◽  
Reaction Rates ◽  
High Energy ◽  
Kinetic Properties ◽  
Catalytic Activities ◽  
Insight Into

We obtained detailed kinetic characteristics–stoichiometry, reaction rates, substrate affinities and equilibrium conditions–of human PPIP5K2 (diphosphoinositol pentakisphosphate kinase 2). This enzyme synthesizes ‘high-energy’ PP-InsPs (diphosphoinositol polyphosphates) by metabolizing InsP6 (inositol hexakisphosphate) and 5-InsP7 (5-diphosphoinositol 1,2,3,4,6-pentakisphosphate) to 1-InsP7 (1-diphosphoinositol 2,3,4,5,6-pentakisphosphate) and InsP8 (1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate), respectively. These data increase our insight into the PPIP5K2 reaction mechanism and clarify the interface between PPIP5K catalytic activities and cellular bioenergetic status. For example, stochiometric analysis uncovered non-productive, substrate-stimulated ATPase activity (thus, approximately 2 and 1.2 ATP molecules are utilized to synthesize each molecule of 1-InsP7 and InsP8, respectively). Impaired ATPase activity of a PPIP5K2-K248A mutant increased atomic-level insight into the enzyme's reaction mechanism. We found PPIP5K2 to be fully reversible as an ATP-synthase in vitro, but our new data contradict previous perceptions that significant ‘reversibility’ occurs in vivo. PPIP5K2 was insensitive to physiological changes in either [AMP] or [ATP]/[ADP] ratios. Those data, together with adenine nucleotide kinetics (ATP Km=20–40 μM), reveal how insulated PPIP5K2 is from cellular bioenergetic challenges. Finally, the specificity constants for PPIP5K2 revise upwards by one-to-two orders of magnitude the inherent catalytic activities of this enzyme, and we show its equilibrium point favours 80–90% depletion of InsP6/5-InsP7.

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