Low Concentrations Of Dimethyl Sulfoxide Research Articles

MOLECULAR INTERACTIONS OF L-TYROSINE IN DIMETHYL SULFOXIDE AQUEOUS SOLUTIONS. PART 1: ELECTRONIC ABSORPTION AND FLUORESCENCE SPECTROSCOPY STUDIES

Molecular interactions of L-tyrosine in dimethyl sulfoxide (DMSO) aqueous solutions have been studied using electronic absorption and fluorescence spectro¬scopy methods. It has been shown that at low concentrations of DMSO in aqueous media hetero-associates such as 1DMSO:1H2O or 1DMSO:2H2O are formed, while at high concentrations, associates of a more complex structure (mixed clusters) are formed that interact with L-tyrosine to form complex compounds.

Determining the Population Doubling Time of HepG2 and Huh-7 Cells and the Toxic Effect of Dimethyl Sulfoxide (DMSO)

Developing cell lines that carry promising qualities closest to human hepatocytes in drug studies is a dream of many research laboratories. About 90% of drugs are metabolised by the liver. Therefore, most drug discovery studies utilise hepatocytes to understand the basic mechanism of the drug’s breakdown. To assure hepatocytes survival, various solvents were tested for their cytotoxicity on the cell line. Since most drugs are weakly soluble in water, they can be dissolved in an aprotic solvent. To obtain accurate results, the requirements of these solvents are biocompatible and non-toxic to the cells. One of the most commonly used solvents is dimethyl sulfoxide (DMSO). This study aims to understand the cell characteristic of cells doubling time and investigate the toxic effect of DMSO using the cell proliferation measurement technique. The toxicity was measured on liver-derived cell lines HepG2 and Huh-7. Cell growth and morphology were observed with an inverted phase microscope while cell viability was counted using Vi-Call XR 2.03. Results showed that a doubling time of 32 hours would be best for treatments on both cells. A concentration of more than 0.40% DMSO has significant toxicity and caused inhibition of proliferation on both cell lines, resulting in low cell count and higher cell death. Ethanol and methanol were observed as good solvents since they have low toxicity. However, drugs that were only dissolved in a low concentration of DMSO (0.05-0.2%) gave the best result without any harmful effect on cell proliferation.

Monitoring Protein Global and Local Parameters in Unfolding and Binding Studies: The Extended Applicability of the Diffusion Coefficient─Molecular Size Empirical Relations.

Protein unfolding and denaturation are main issues in biochemical and pharmaceutical research. Using a global parameter, the translational diffusion coefficient D, folded, unfolded, and intrinsically disordered proteins of a given molar mass M can be distinguished based on their distinct hydrodynamic properties. For broader applications, we provide generalized, PFG-NMR-based empirical D-M relations validated at different temperatures and ready to use with the corresponding corrections in different media. We demonstrate that these relations enable a more accurate molecular mass determination and show fewer potential errors than those of the common methods based on small-molecular diffusion standards. We monitor unfolding of three model proteins using 8 M urea and dimethyl sulfoxide (DMSO)-water mixtures as denaturing agents, highlighting the effect of disulfide bonds. Denaturation in 8 M urea is pH-dependent; in addition, for proteins with highly stable disulfide bonds, a reducing agent (TCEP) is required to achieve complete unfolding. Regarding the effect of local parameters, we show that at low DMSO concentrations─common conditions in pharmaceutical binding studies─the PFG-NMR-derived global parameters are not significantly affected. Still, the atomic environments can change, and the bound solvent molecule can inhibit the binding of a partner molecule. Using proteins with natural isotopic abundance, this effect can be proven by fast 1H-15N 2D correlation spectra. Our results enable fast and easy estimation of protein molecular mass and the degree of folding in various media; moreover, the effect of the cosolvent on the atomic-level structure can be traced without the need of isotope labeling.

Low dose DMSO treatment induces oligomerization and accelerates aggregation of \u03b1-synuclein

Dimethyl sulfoxide (DMSO) is a highly utilized small molecule that serves many purposes in scientific research. DMSO offers unique polar, aprotic and amphiphilic features, which makes it an ideal solvent for a wide variety of both polar and nonpolar molecules. Furthermore, DMSO is often used as a cryoprotectant in cell-based research. However, recent reports suggest that DMSO, even at low concentration, might interfere with important cellular processes, and cause macromolecular changes to proteins where a shift from α-helical to β-sheet structure can be observed. To investigate how DMSO might influence current research, we assessed biochemical and cellular impacts of DMSO treatment on the structure of the aggregation-prone protein α-synuclein, which plays a central role in the etiology of Parkinson’s disease, and other brain-related disorders, collectively termed the synucleinopathies. Here, we found that addition of DMSO increased the particle-size of α-synuclein, and accelerated the formation of seeding-potent fibrils in a dose-dependent manner. These fibrils made in the presence of DMSO were indistinguishable from fibrils made in pure PBS, when assessed by proteolytic digestion, cytotoxic profile and their ability to seed cellular aggregation of α-synuclein. Moreover, as evident through binding to the MJFR-14-6-4-2 antibody, which preferentially recognizes aggregated forms of α-synuclein, and a bimolecular fluorescence complementation assay, cells exposed to DMSO experienced increased aggregation of α-synuclein. However, no observable α-synuclein abnormalities nor differences in neuronal survival were detected after oral DMSO-treatment in either C57BL/6- or α-synuclein transgenic F28 mice. In summary, we demonstrate that low concentrations of DMSO makes α-synuclein susceptible to undergo aggregation both in vitro and in cells. This may affect experimental outcomes when studying α-synuclein in the presence of DMSO, and should call for careful consideration when such experiments are planned.

On crystallization of water confined in liposomes and cryoprotective action of DMSO.

In this work, the phase behavior of cryoprotective mixtures based on dimethyl sulfoxide (DMSO) mixed with a lipid bilayer consisting of dimyristoyl phosphatidylcholine (DMPC) was studied. This system represented a model of a biological cell and its membrane. The aim of the work was to clarify the origin of the cryoprotective action of low-concentrated mixtures (1–10 vol%) DMSO in water, representing mixtures used in cryopreservation in cell therapy. The combination of experimental techniques of differential scanning calorimetry (DSC) and positron annihilation lifetime spectroscopy (PALS) allowed a study of crystallization behavior of water confined in liposomes imitating the intracellular environment. The ability of liposomes to show the fundamental aspects of water phase behavior seen during freezing of biological cells was proved. The presence of an amorphous freeze-concentrated phase of DMSO in the frozen state was confirmed and its possible crystallization into the DMSO trihydrate and ice during thawing was demonstrated. Correlation between the critical temperature range for the loss of cell viability during slow thawing and the temperatures of freeze-concentrated phase crystallization was found. Based on this finding, possible mechanisms of DMSO cryoprotection are discussed with support brought by results for the studied model system. Quantification of the ice phase fraction in the frozen mixtures revealed that even low concentrations of DMSO can induce a considerable decrease in the amount of ice present.

Enhanced Cellular Cryopreservation by Biopolymer-Associated Suppression of RhoA/ROCK Signaling Pathway.

With increasing demands on long-term storage of cells, cryopreservation of cells is gaining more importance in cell-based research and applications. Dimethyl sulfoxide (DMSO) is a commonly used chemical cryoprotectant, providing increased cell survival during the freezing process. However, its use is limited in clinical applications due to its low biocompatibility above cryogenic temperatures. Herein, we present a new approach for reducing the use of DMSO in cryopreservation by using biodegradable hyaluronic acids (HAs). By adding HAs into cryoprotectant media containing a low concentration of DMSO, higher cell viability and cell proliferation rate were observed upon thawing after cryopreservation. The HA-supplemented cryopreservation media did not reduce the size of the ice crystal, which significantly influenced cell viability during cell freezing, but decreased the Ras homolog family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling pathway related to apoptosis. The cell-interactive cryoprotectants containing HA can be applied to the development of a new cryoprotectant that reduces the adverse effect of DMSO.

Study on Hemostimulating Properties of Granulocyte-Macrophage Colony Stimulating Factor

The hemostimulating properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) make possible its clinical use in alleviating side effects of anti-cancer radio- and chemotherapy, in bone marrow transplantation, and in the treatment of some primary immunodeficiency conditions associated with leukopenia. The State Research Center of Virology and Biotechnology “Vector” of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing has developed a high-performance technology for production of recombinant human GM-CSF (rhGM-CSF) based on a recombinant E. coli strain.The aim of the study was to assess hemostimulating activity of the rhGM-CSF preparation obtained using the new developed technology, as observed in cell culture and in the mice model of myelosuppression induced by cyclophosphamide administration.Materials and methods: in vitro evaluation of rhGM-CSF hemostimulating activity was performed by MTT assay in the commercial HL-60 promyelocytic leukemia cell culture with preliminary suppression of cell growth rate by adding a low concentration of dimethyl sulfoxide to the medium. In vivo studies were carried out in CBA/CaLac mice with cyclophosphamide-induced myelosuppression. The hemostimulating properties of the drug were evaluated after subcutaneous administration of 1–175 µg/kg doses for 4–5 days, following administration of a cytostatic agent. The total number of leukocytes and the content of their morphological forms were determined in blood samples taken at different time points after the drug administration. The statistical processing of the experimental data was based on analysis of variance using Statgraphics v. 5.0 software.Results: the proliferative activity of HL-60 cells incubated with the rhGM-CSF preparation for 72 hours was shown to be dose-dependent. The highest values of the increase in proliferative activity associated with an increase in the drug dose were observed in the concentration range from 0.04 to 0.64 ng/mL (proliferative activity increased by 11–18% when the dose was increased twofold). The experiments in mice demonstrated a two-phase pattern of the dose-dependent effect. The drug showed the highest hemostimulating effect at the dose of 90 µg/kg.Conclusions: the rhGM-CSF preparation obtained using the new developed technology has a pronounced hemostimulating activity confirmed by both in vitro and in vivo test systems.

Comparative cytotoxic effects of methanol, ethanol and DMSO on human cancer cell lines

Introduction: Anti-tumor drug screening is the most popular technique in drug discovery. In this technique, various agents were tested for their cytotoxicity on cell lines, particularly cancer cell lines. Since most of these agents are weakly soluble in water, they can be normally dissolved in lipophilic solvents. To obtain accurate results, the requirement of these solvents are that they be biocompatible and non-toxic to cells. The aim of this study was to investigate the biological effects of the three agents most commonly used as drug vehicles, i.e. dimethyl sulfoxide (DMSO), ethanol and methanol, using cell proliferation measurement techniques.
 Methods: To minimize the errors from the measurement techniques, this study used the xCelligence RTCA system which entails real-time monitoring of cell proliferation without dye labeling. Under the wide range concentration of solvents (from 0% to 100% of solvents by serial dilutions), the cell index (CI) and slope of cell proliferation curves of HepG2, MDA-MB-231, MCF-7 and VNBRCA1 cell lines were analyzed (by the software provided by the xCelligence system).
 Results: The results showed that DMSO had a significant toxicity and inhibition of proliferation on 4 cell lines, at the concentrations of 10%, 5%, 2.5% and 1.25% (p<0.0001). Methanol and ethanol inhibited cellular proliferation at the concentrations of 10% and 5% (p<0.0001). The concentrations of ethanol and methanol ranging from 2.5% to 0.15% concentration were well-tolerated by cells with respect to proliferation. Depending on the extracts or agents, each should be diluted in the suitable vehicle at the proper concentrations.
 Conclusion: Ethanol and methanol are good choices for solvents since they have low toxicity on HepG2, MDA-MB-231, MCF-7 and VNBRCA1 cell lines. However, in the case of agents only dissolvable in DMSO, low concentrations of DMSO from 0.6%-0.015% should be considered.

Ultrafast Dynamics at the Lipid-Water Interface: DMSO Modulates H-Bond Lifetimes.

Dimethyl sulfoxide (DMSO) is a common cosolvent and cryopreservation agent used to freeze cells and tissues. DMSO alters the H-bond structure of water, but its interactions with biomolecules and, specifically, with biological interfaces remain poorly understood. Here we investigate the effects of DMSO on the H-bond dynamics at the lipid-water interface using a combination of ultrafast two-dimensional infrared (2D IR) spectroscopy and molecular dynamics simulations. Ester carbonyl absorption spectra show that DMSO dehydrates the interface, and simulations show that the area per lipid is decreased. Ultrafast 2D IR spectra measure the time scales of frequency fluctuations at the ester carbonyl positions located precisely between the hydrophobic and hydrophilic regions of the membrane. 2D IR measurements show that low DMSO concentrations (<10 mol %) induce ∼40% faster H-bond dynamics compared with pure water, whereas increased concentrations (>10-20 mol %) once again slow down the dynamics. This slow-fast-slow trend is described in terms of two different solvation regimes. Below 10 mol %, DMSO weakens the interfacial H bond, leading to faster "bulk-like" dynamics, whereas above 10 mol %, water molecules become "relatively immobilized" as the H-bond networks becoming disrupted by the H-bond donor/acceptor imbalance at the interface. These studies are an important step toward characterizing the environments around lipid membranes, which are essential to numerous biological processes.

Cryoprotectants Disrupt Hydrogen-Bond Networks at the Lipid-Water Interface

Cryoprotectant agents (CPAs) are mixtures of small organic compounds used to inhibit ice-crystal growth during cell and tissue freezing and storage, but these compounds can be themselves toxic to cells. Increased membrane permeability, caused by partial disruption of lipid-lipid interactions, combined with dehydration, have been hypothesized to be the main driver of cell toxicity, but the specific interactions between lipids and cryoprotectants are poorly understood. In this study, we investigate the effects of dimethyl sulfoxide (DMSO) on phosphatidylcholine bilayers. We have measured ultrafast two-dimensional infrared (2D IR) spectra of the ester carbonyl vibrations to directly probe the interfacial hydrogen bond networks. Our measurements directly quantify the picosecond hydrogen bond lifetimes at the ester carbonyl positions. Spectra are interpreted at an atomistic level using molecular dynamics simulations, which are in semi-quantitative agreement with experiment. Our results show that biologically relevant cryoprotectant concentrations (<20 mol%), partially dehydrate the interface, but surprisingly, hydrogen-bond lifetimes are non-monotonic with increasing concentration: 1. Low DMSO concentrations (<10%) severely disrupt the otherwise ice-like interfacial hydrogen bond networks, without altering the lipid-lipid interactions, leading to faster overall hydrogen bond dynamics at the ester carbonyls. 2. Above >10 mol%, dynamics become slower as a result of dehydration, more rigid H-bond conformations, trapped water, and stronger headgroup-headgroup interactions. Together, these results provide an atomistic foundation for understanding the complex effects of cryoprotectants on lipid membranes, showing that different effects dictate the properties of membranes within various cryoprotectant concentration regimes.

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Influence of polar solvents on permeability, stiffness and collagen dissociation of demineralized dentin

The purpose of this in vitro study was to evaluate and compare the effect of dimethyl sulfoxide (DMSO) or ethanol on the permeability, stiffness and collagen dissociation of demineralized dentin.Dentin cubes (2 × 2 × 2 mm) were demineralized in EDTA and incubated in DMSO or ethanol (0.01, 0.1, 1, 5, 10, 20, 50 and 100%) (n= 10/group) for 30 s, followed by 100% HEMA incubation. Extracted HEMA was quantified. For elastic modulus (E), demineralized dentin beams (6 × 2 × 1 mm) were incubated in DMSO or ethanol (1, 10, 20, 50 and 100%) for 10, 30 or 60 min at 3-point bending. Additional demineralized dentin discs (1 mm) were incubated in DMSO (1, 10, 50 and 100%) for 10, 30 and 60 min and the optical clearing effect was observed. The data were analyzed using ANOVA and Tukey's test (α=0.05) using SigmaPlot (Systat Software Inc., San Jose, CA).Compared to controls, HEMA uptake was significantly higher with all DMSO concentrations, and with 0.1% or higher ethanol concentrations (p<0.05). HEMA uptake in DMSO-incubated specimens (0.01, 5 and 10%) was significantly higher than with the ethanol incubation. Significant increase in elastic moduli was observed with 50–100% DMSO- and only with 100% ethanol after 10 min incubation. The optical clearing effect of 50–100% DMSO-incubated dentin disks was observed starting from 10 min.The pretreatment of dentin surfaces with low concentrations of DMSO resulted in significant improvement of the penetration of monomers to demineralized dentin matrices. Increase in penetration of monomers combined with a reversible stiffening of dentin collagenous matrix may explain the previously shown increase in durability of wet- or dry-bonded adhesive interfaces with DMSO treatment.

CD4+ T cell activation, function, and metabolism are inhibited by low concentrations of DMSO

Dimethyl sulfoxide (DMSO) is a polar organic solvent used in a wide range of biological applications. DMSO is routinely used as a cryoprotectant for long-term cell freezing as well as to dissolve peptides and drugs for immune cell functional assays. Here, human CD4+ T cell activation, cytokine production, proliferation, and metabolism were investigated after stimulation in the presence of 0.01% to 1%, DMSO, representing concentrations commonly used in vitro. Surface expression of the activation markers CD69, CD25 and CD154 after polyclonal activation of CD4+ T cells was inhibited by 0.25% or higher concentrations of DMSO. The frequencies of IL-21+, IL-4+, and IL-22+ CD4+ T cells, following polyclonal activation were variably inhibited by DMSO at concentrations ranging from 0.25% to 1%, whereas IFNγ+ cells were unaffected. CD4+ T cell proliferation after anti-CD3 or antigen stimulation was inhibited by 0.5% DMSO and abolished by 1% DMSO. After polyclonal stimulation, glucose uptake was inhibited in the presence of 1% DMSO, but only minor effects on CD4+ T cell respiration were observed. Consistent with the immune effects, the gene expression of early signaling and activation pathways were inhibited in CD4+ T cells in the presence of 1% DMSO. Our study revealed that DMSO at concentrations generally used for in vitro studies of T cells impacts multiple features of T cell function. Therefore, we urge care when adding DMSO-containing preparations to T cell cultures.

Molecular Self-Diffusion Coefficients in Solutions of Dimethylsulfoxide in Monoethanolamine

The molecular self-diffusion coefficients of dimethylsulfoxide (DMSO) in monoethanolamine (MEA) are measured by means of proton NMR spin-echo spectroscopy at 304.6 K and DMSO concentrations varying from 0 to 30 mol %. The drop in the self-diffusion coefficient of MEA observed at low DMSO concentrations is attributed to the solvophobic effect DMSO molecules exhibit in solutions in MEA. The results obtained for the considered system are compared to similar data for the DMSO–ethylene glycol system. The differences between the two systems are explained by the higher lability of spatial hydrogen bond networks in liquid MEA, compared to ethylene glycol.

Hydrogen-Bonding Interactions in Pyridinium-Based Ionic Liquids and Dimethyl Sulfoxide Binary Systems: A Combined Experimental and Computational Study.

The addition of highly polar and aprotic cosolvents to ionic liquids has proven to considerably decrease the viscosity of the solution and improve mass transfer in many chemical reactions. In this work, the interactions between a representative pyridinium-based ionic liquid, N-butylpyridinium dicyanamide ([Bpy][DCA]), and a cosolvent, dimethylsulfoxide (DMSO), were studied in detail by the combined use of attenuated total reflection Fourier transform infrared spectroscopy, hydrogen nuclear magnetic resonance (1H NMR), and density functional theory calculations. Several species in the [Bpy][DCA]–DMSO mixtures have been identified, that is, ion clusters can translate into ion pairs during the dilution process. DMSO formed hydrogen bonds (H bonds) simultaneously with [Bpy]+ cations and [DCA]− anions but stronger hydrogen-bonding interactions with the [Bpy]+ cations than the [DCA]− anions, and the intrinsic hydrogen-bond networks of IL were difficult to interrupt at low DMSO concentrations. Interestingly, hydrogen-bonding interactions reach the strongest when the molar fraction of DMSO is 0.4–0.5. Hydrogen-bonding interactions are prominent in the chemical shifts of hydrogen atoms in [Bpy]+ cations, and anisotropy is the main reason for the upfield shifts of DMSO in the presence of [Bpy][DCA]. The theoretical calculations offer in-depth studies of the structural evolution and NMR calculation.

Trehalose and ascorbic acid improves the Cryopreservation of umbilical Cord Blood hematopoietic stem Cells (CD34+) with low Concentrations of Dimethylsulfoxide

Cryopreservation of umbilical cord hematopoietic stem cells (HSCs) is essential step in stem cell transplantation. The Dimethylsulfoxide (DMSO) mostly used as a cryoprotective agent, associated with toxic effects to stem cells. In order to minimize the effects of DMSO, low concentrations of DMSO with additional cryoprotectants should used. In this study mononuclear cells (MNCs) isolated by ficoll from umbilical cord blood (UCB), which contain hematopoietic stem cells (CD34+), were used for cryopreservation. Cryopreservation of UCB-derived MNCs was done for period of one month by using uncontrolled-rate freezing technique at -196˚C in liquid nitrogen. Cryopreservation solution was used which consisted of minimum essential medium (MEM) and 20% fetal calf serum (FCS) supplemented with the cryoprotectant dimethylsulfoxide (DMSO) in two concentrations 2.5% and 5% DMSO, lower than usually used 10%, with and without 25µg/ml trehalose or 80µg/ml ascorbic acid to improve cryopreservation process. The addition of trehalose and ascorbic acid improved cryopreservation process in comparison with control. Addition of 5% DMSO alone and with additives showed a better result than 2.5% DMSO alone or with additives for cryopreserving CD34+ cells as indicated by immunocytochemistry. Washing out DMSO also affected the count and viability of MNCs. These results indicated that it could use low concentrations of DMSO in cryopreservation of HSCs by association with trehalose and ascorbic acid.

Probing the replacement of water by dimethyl sulfoxide in the hydration shell of N-methylacetamide by FTIR-spectroscopy

The influence of dimethyl sulfoxide (DMSO) on the hydration of a peptide group has been explored by measuring the FTIR spectrum of N-methylacetamide in different mixtures of DMSO and D2O. An analysis of the amide I’ profile indicates two processes. For DMSO mole fractions between 0 and 0.5, the hydration shell on the side of the carbonyl group is systematically perturbed, which leads to a destabilization and eventual elimination of the hydrogen bonding between the carbonyl group and D2O. Upon further increasing the mole fraction of DMSO, water on the amide group side of the peptide is completely replaced by DMSO and the NH⇋ND exchange is significantly impeded. In mixtures with a very high fraction of DMSO, NMA with and without or weak hydrogen bonding to a DMSO molecule coexist with the latter configuration being clearly dominant in pure DMSO. Our results corroborate the recently debated notion that DMSO functions as a chaotropic cosolvent even at low fractions in that it exhibits preferential binding of water to the solute and by perturbing the stability of the peptide-water entity. They also reveal a rather fast exchange between different peptide-solvent complexes at low DMSO concentrations.

Probing the emitter site of Renilla luciferase using small organic molecules; an attempt to understand the molecular architecture of the emitter site

Renilla luciferase is a sensitive enzyme and has wide applications in biotechnology such as drug screening. Previous studies have tried to show the catalytic residues, nevertheless, the accurate architecture and molecular behavior of its emitter site remains uncharacterized. In this study, the activity of Renilla luciferase, in the presence of two small organic molecules including dimethyl sulfoxide (DMSO) and isopropanol was considered and the structure was studied by circular dichroism (CD) and fluorescence spectroscopy. Moreover, the interaction of small organic molecules with the Renilla luciferase was studied using molecular dynamics simulations. Kinetics studies showed that at low concentration of DMSO (16.6–66mM) and isopropanol (19.3–76mM) the Km changed and a competitive inhibition pattern was observed. Moreover, spectroscopy studies reveled that the changes of activity of Renilla luciferase in the presence of low concentrations of small organic molecules was not associated with structural collapse or severe changes in the enzyme conformation. Molecular dynamics simulations indicated that DMSO and isopropanol, as probing molecules, were both able to bind to the emitter site and remained with the residues of the emitter site. Based on the probing data, the architecture of the emitter site in the “non-binding” model was proposed.

Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation.

Background:L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation.Methods:The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG), comparing with the control group (15% DMSO and 15% EG without L-proline). The survival rate, 5-methylcytosine (5-mC) expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test.Results:L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control.Conclusions:It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification.

Protein Structural Memory Influences Ligand Binding Mode(s) and Unbinding Rates.

The binding of small molecules (e.g., natural ligands, metabolites, and drugs) to proteins governs most biochemical pathways and physiological processes. Here, we use molecular dynamics to investigate the unbinding of dimethyl sulfoxide (DMSO) from two distinct states of a small rotamase enzyme, the FK506-binding protein (FKBP). These states correspond to the FKBP protein relaxed with and without DMSO in the active site. Since the time scale of ligand unbinding (2-20 ns) is faster than protein relaxation (100 ns), a novel methodology is introduced to relax the protein without having to introduce an artificial constraint. The simulation results show that the unbinding time is an order of magnitude longer for dissociation from the DMSO-bound state (holo-relaxed). That is, the actual rate of unbinding depends on the state of the protein, with the protein having a long-lived memory. The rate thus depends on the concentration of the ligand as the apo and holo states reflect low and high concentrations of DMSO, respectively. Moreover, there are multiple binding modes in the apo-relaxed state, while a single binding mode dominates the holo-relaxed state in which DMSO acts as hydrogen bond acceptor from the backbone NH of Ile56, as in the crystal structure of the DMSO/FKBP complex. The solvent relaxes very fast (∼1 ns) close to the NH of Ile56 and with the same time scale of the protein far away from the active site. These results have implications for high-throughput docking, which makes use of a rigid structure of the protein target.