Absence Of Dimethyl Sulfoxide Research Articles

Acetone-induced structural variant of insulin amyloid fibrils

Self-propagating polymorphism of amyloid fibrils is a distinct manifestation of non-equilibrium conditions under which protein aggregation typically occurs. Structural variants of fibrils can often be accessed through physicochemical perturbations of the de novo aggregation process. On the other hand, tiny changes in the amino acid sequence of the parent protein may also result in structurally distinguishable amyloid fibrils. Here, we show that in the presence of acetone, the low-pH fibrillization pathway of bovine insulin (BI) leads to a new type of amyloid with the infrared features (split amide I′ band with the maximum at 1623 cm−1) bearing a striking resemblance to those of the previously reported fibrils from recombinant LysB31-ArgB32 human insulin analog formed in the absence of the co-solvent. Insulin fibrils formed in the presence ([BI-ace]) and absence ([BI]) of acetone cross-seed each other and pass their infrared features to the daughter generations of fibrils. We have used dimethyl sulfoxide (DMSO) coupled to in situ infrared spectroscopy measurements to probe the stability of fibrils against chemical denaturation. While both types of fibrils eventually undergo DMSO-induced disassembly coupled to a β-sheet→coil transition, in the case of [BI-ace] amyloid, the denaturation is preceded by the fibrils transiently acquiring the [BI]-like infrared characteristics. We argue that this effect is caused by DMSO-induced dehydration of [BI-ace]. In support to this hypothesis, we show that, even in the absence of DMSO, the infrared features of [BI-ace] disappear upon drying. We discuss this very peculiar aspect of [BI-ace] fibrils in the context of recently accessed in silico models of plausible structural variants of insulin protofilaments.

Photodegradation of free base and zinc porphyrins in the presence and absence of oxygen

Comparison of photostability in degassed and aerated toluene solutions is reported for 5,10,15,20-tetraphenylporphyrin, 5,10,15-tri(p-tolyl)porphyrin, and their zinc analogues. After degassing, quantum yields of photodegradation are higher, but the photodecomposition rates decrease. Lower stability in deoxygenated solutions is due to much longer triplet lifetimes: 200–300 microseconds, compared to 200–360 ns in non-degassed toluene. For the zinc porphyrins, the LC–MS results show that the initial photoproduct contains two oxygen atoms. Based on electronic absorption and calculations, it is assigned to dehydrated zinc biladienone structure, relatively stable in toluene, but readily demetallated in dichloromethane. A similar species is formed also in the case of free bases, but it then undergoes hydration due to traces of water present in the solvent. Zinc derivatives were found to form biladienones even in degassed solutions. To explain this observation, we postulate formation of a complex with remaining oxygen or oxygen-containing species which is not removed by freeze–thaw procedure. This hypothesis is confirmed by MS results and by the analysis of photodegradation products obtained when zinc porphyrin is complexed with dimethylsulfoxide (DMSO). Under these circumstances, changes in absorption are the same as in the absence of DMSO when non-degassed toluene is used, but irradiation of deoxygenated solutions leads to a different photoproduct. For both degassed and non-degassed solvents, complexation with DMSO results in the enhancement of photostability.Graphical abstract

Tuning the course of the oxygen reduction reaction at a carbon electrode using alkaline electrolytes based on binary DMSO–water solvent mixtures

We studied the oxygen reduction reaction (ORR) at a glassy carbon electrode in KOH electrolytes based on DMSO–water solvent mixtures of various compositions (ranging from 0.0 to 93.3 vol% DMSO). The results of RDE (rotating disk electrode) experiments show that by changing the composition ofthe DMSO–water solvent mixture, one can control the course of oxygen electroreduction. Specifically, in the absence of dimethyl sulfoxide, the ORR at −1.5 V vs. Hg/HgO proceeds mainly as a two-electron process (n = 2.28 at 400 rpm), whereas with increasing DMSO content the ORR shifts gradually towards complete four-electron reduction. This tendency continues until the maximum electron transfer number is reached (n = 3.70 at 400 rpm) in an alkaline electrolyte based on a 1:1 H2O:DMSO (v/v) solvent mixture. The effect of DMSO addition on the course of the ORR process at the carbon electrode is thought to arise from the unique physicochemical properties of DMSO–water systems, i.e. their high viscosity and polarity as a result of H-bonded intermolecular complexes formed between water and DMSO molecules.

Oxygen Plasma Substrate and Specific Nanopattern Promote Early Differentiation of HepaRG Progenitors.

Fully differentiated HepaRG™ cells are the hepatic cell line of choice for in vitro study in toxicology and drug trials. They are derived from a hepatoblast-like progenitor (HepaRG-P) that differentiates into a coculture of hepatocyte-like and cholangiocyte-like cells. This process that requires 2 weeks of proliferation followed by 2 weeks of differentiation using dimethyl sulfoxide (DMSO) can be time consuming and costly. Identifying a method to accelerate HepaRG-Ps toward a mature lineage would save both time and money. The ability to do this in the absence of DMSO would remove the possibility of confounding toxicology results caused by DMSO induction of CYP pathways. It has been shown that tissue culture substrates play an important role in the development and maturity of a cell line, and this is particularly important for progenitor cells, which retain some form of plasticity. Oxygen plasma treatment is used extensively to modify cell culture substrates. There is also evidence that patterned rather than planar surfaces have a positive effect on proliferation and differentiation. In this study, we compared the effect of standard tissue culture plastic (TCP), oxygen plasma coated (OPC), and nanopatterned substrates (NPS) on early differentiation and function of HepaRG-P cells. Since NPS were OPC we initially compared the effect of TCP and OPC to enable comparison between all three culture surfaces using OPC as control to asses if patterning further enhanced early differentiation and functionality. The results show that HepaRG-P's grown on OPC substrate exhibited earlier differentiation, proliferation, and function compared with TCP. Culturing HepaRG-P's on OPC with the addition of NPS did not confer any additional advantage. In conclusion, OPC surface appeared to enhance hepatic differentiation and functionality and could replace traditional methods of differentiating HepaRG-P cells into fully differentiated and functional HepaRGs earlier than standard methods. Impact statement We show significantly earlier differentiation and function of HepaRG progenitor cells when grown in dimethyl sulfoxide-free medium on oxygen plasma substrates versus standard tissue culture plastic. Further investigation showed that nanopatterning of oxygen plasma substrates did not confer any additional advantage over smooth oxygen plasma, although one pattern (DSQ120) showed comparable early differentiation and function.

Cryobiological parameters of multipotent stromal cells obtained from different sources

Stem cells are important for regenerative medicine mainly due to their multilineage differentiation capacity. However, the cells rapidly loose this capability during culturing. Cryopreservation preserves the differentiation potential of the cells, until they are needed. In this study, specific cell properties of multipotent stromal cells (MSCs), from the common marmoset monkey Callithrix jacchus MSCs derived from amnion (Am) and bone marrow (Bm) were studied in order to predict optimal cooling rates for cryopreservation. Cell volume behaviour in anisotonic media, hydraulic membrane permeability at supra as well as subzero temperatures, and time point of intracellular ice formation (IIF) were investigated by Coulter Counter and cryomicroscopy. Cryopreservation outcome was studied using the predicted and experimentally determined cooling rate followed by 24 h re-cultivation. Little differences in osmotically inactive volume were found between amnion (0.27 × Vo) and bone marrow (0.28 × Vo) derived MSCs. The activation energy for water transport at suprazero temperature was found to be similar for both cell types; 4.4 ± 0.2 and 5.0 ± 0.15 kcal mol−1 for amnion and bone marrow derived MSCs, respectively. At subzero temperatures in the absence of dimethyl sulfoxide (Me2SO), the activation energy for water transport increased to 24.8 ± 3 kcal mol−1 and 27.4 ± 0.9 kcal mol−1 for Am and BmMSCs respectively. In the presence of Me2SO, activation energies were found to be 11.6 ± 0.3 kcal mol−1 and 19.5 ± 0.5 kcal mol−1 respectively. Furthermore, Me2SO was found to decrease the incidence of intracellular ice formation. The predicted optimal cooling rates of 11.6 ± 0.9 °C/min (AmMSCs) and 16.3 ± 0.5 °C/min (BmMSCs) resulted in similar post-thaw viability values compared to the experimentally determined optimal cooling profiles of 7.5 °C/min to −30 °C, followed by 3 °C/min to −80 °C.

Crystal Structures of the Amide and Iminol Tautomers O-(3,5-dinitrobenzoyloxy)benzohydroxamate. A Case of a Disappearing Solvate?

The O-(3,5-dinitrobenzoyl) derivative of benzohydroxamic acid 1 crystallizes in its iminol tautomeric form, hydrogen bonded to a molecule of dimethyl sulfoxide (DMSO) when crystallized from methanol in the presence of DMSO. In contrast, in the absence of DMSO, 1 crystallizes in its amide tautomeric form. Computations suggest that in the absence of DMSO, the amide tautomer of 1 is significantly more stable than the iminol form. Conversely, in the presence of DMSO, the two forms have comparable stabilities. Solution infrared spectra of 1 run in dichloromethane are consistent with the amide tautomeric form predominating; however, titration of aliquots of DMSO results in the formation of a second species with spectral characteristics consistent with the iminol tautomer.

Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. Stem Cells 2015;33:1187–1199

C-2018: Human umbilical vein endothelial cell (HUVEC) response to interrupted cooling in the presence and absence of dimethyl sulfoxide

HUVECs were first successfully cultured in vitro in 1973, initiating the growth of modern vascular biology. Study into HUVEC cryopreservation has made it readily available for purchase, however the cryobiological response of HUVECs has not been fully investigated. Viability by membrane integrity of HUVECs as received from the supplier was 50 ± 2%, indicating that cryopreservation of HUVECs can be improved. Membrane hydraulic conductivity is an osmotic property which is necessary to simulate cryobiological response. HUVEC membrane hydraulic conductivity was measured and found to be 0.147 μm 3 /μm 2 /min/atm at 20 °C, less than fibroblasts and TF-1 cells, therefore being the slowest osmotically-responding cells studied using interrupted cooling. HUVECs were subjected to interrupted slow cooling without hold time (graded freezing) and rapid cooling interrupted with a hold time (two-step freezing) in the presence or absence of 10% Me 2 SO. Preliminary results show that HUVECs have higher viabilities from graded freezing than from two-step freezing, similar to previously studied fibroblasts but different from TF-1 cells. Optimal cooling procedures cannot solely be explained by hydraulic conductivity. Graded freezing at 1 °C/min in the presence of 10% Me 2 SO resulted in the best viability, with almost no damage from direct thaw after cooling to between −5 °C and −40 °C and a maximum of 66% HUVEC membrane integrity after cooling to −35 °C, plunging into liquid nitrogen and then thawing (plunge-thaw). Two-step freezing in the presence of 10% Me 2 SO resulted in almost no damage from direct thaw from as low as −15 °C. Lower experimental hold temperatures resulted in damage after direct thaw and the highest membrane integrity after plunge-thaw was 42% for a hold temperature of −25 °C. Interrupted cooling in the absence of Me 2 SO resulted in no HUVEC membrane integrity after plunge-thaw. The understanding gained from this investigation may lead to improved cryopreservation of HUVECs. Funding: CIHR.

94 The investigation of Hoechst 33258 interaction with DNA in aqueous solutions of dimethylsulfoxide

The melting of DNA complexes with Hoechst 33258 (H33258) has been carried out in both presence and absence of dimethylsulfoxide (DMSO) to reveal the effect of the mentioned compound on melting parameters of DNA-H33258 complexes at r = 0.1, where r = [H33258]/[DNA] and at 0.02 M Na+ ion concentration. It is known that the structural–functional changes of macromolecules take place in aqueous solutions of DMSO (Markarian et al., 2006). It has been shown earlier that DNA hydration degree increase results in sharp changing of the binding mechanism of H33258 to DNA: AT-specific mode of binding disappears and ligand molecules start to intercalate into DNA at r ⩽ 0.1 (Vardevanyan et al., 2008). Moreover, the increase of T m of complexes is higher compared to the case of relatively low hydration degree of DNA. This is conditioned by the fact that at DNA hydration, increasing the intercalation becomes more preferable since in this case hydrophobic ligand molecules are shielded from water more effectively. The results obtained confirm that with the increasing of DMSO content in solution the melting temperature, T m, decreases. Moreover, denaturation of complexes leads to the decrease of the fluorescence intensity of ligand molecule. We suggest that the observed phenomenon happens due to the structural change of solution which promotes dissociation of ligand molecules bound to DNA, and hence a facilitated denaturation of DNA-H33258 complexes occurs.

Phase 1 and Phase 2 Drug Metabolism and Bile Acid Production of HepaRG Cells in a Bioartificial Liver in Absence of Dimethyl Sulfoxide

The human liver cell line HepaRG has been recognized as a promising source for in vitro testing of metabolism and toxicity of compounds. However, currently the hepatic differentiation of these cells relies on exposure to dimethylsulfoxide (DMSO), which, as a side effect, has a cytotoxic effect and represses an all-round hepatic functionality. The AMC-bioartificial liver (AMC-BAL) is a three-dimensional bioreactor that has previously been shown to upregulate various liver functions of cultured cells. We therefore cultured HepaRG cells in the AMC-BAL without DMSO and characterized the drug metabolism. Within 14 days of culture, the HepaRG-AMC-BALs contained highly polarized viable liver-like tissue with heterogeneous expression of CYP3A4. We found a substantial metabolism of the tested substrates, ranging from 26% (UDP-glucuronosyltransferase 1A1), 47% (CYP3A4), to 240% (CYP2C9) of primary human hepatocytes. The CYP3A4 activity could be induced 2-fold by rifampicin, whereas CYP2C9 activity remained equally high. The HepaRG-AMC-BAL secreted bile acids at 43% the rate of primary human hepatocytes and demonstrated hydroxylation, conjugation, and transport of bile salts. Concluding, culturing HepaRG cells in the AMC-BAL yields substantial phase 1 and phase 2 drug metabolism, while maintaining high viability, rendering DMSO addition superfluous for the promotion of drug metabolism. Therefore, AMC-BAL culturing makes the HepaRG cells more suitable for testing metabolism and toxicity of drugs.

Effect of distance between decaying 125I and DNA on Auger-electron induced double-strand break yield

Purpose: To determine the possible effects of 125I-to-DNA distance on the magnitude and mechanism of Auger-electron induced-double-strand break (DSB) production.Materials and methods: We have synthesized a series of 125I-labeled Hoechst (H) derivatives (125IE–H, 125IB–H, 125I-C8–H and 125I-C12–H). While all four molecules share a common DNA minor groove binding bis-benzimidazole motif, they are designed to position 125I at varying distances from the DNA helix. Each Hoechst derivative was incubated at 4°C in phosphate buffered saline (PBS) together with supercoiled (SC) 3H-pUC19 plasmid DNA (ratio 3:1) ± the •OH scavenger dimethyl sulfoxide (DMSO) (0.2 M). Aliquots were analyzed on agarose gels over time and DSB yields per decay of 125I atom were determined. Docking of the iodinated compounds on a DNA molecule was carried out to determine the distance between the iodine atom and the central axis of DNA.Results: In the absence of DMSO, the results show that the DSB yields decrease monotonically as the 125I atom is distanced – by 10.5 Å to 13.9 Å – from the DNA helix (125IEH: 0.52 ± 0.01; 125IB–H: 0.24 ± 0.03; 125I-C8–H: 0.18 ± 0.02; 125I-C12–H: 0.10 ± 0.00). In the presence of DMSO, DSB yields for 125IEH (0.49 ± 0.02) and 125IB-H (0.26 ± 0.04) remain largely unchanged indicating that DSB are entirely produced by direct effects. Strikingly, 125I-C8–H or 125I-C12–H, did not produce detectable DSB in the presence of DMSO under similar conditions suggesting when 125I atom is positioned > 12 Å from the DNA, DSB are entirely produced by indirect effects.Conclusion: These results suggest that at a critical distance between the 125I atom and the DNA helix, DSB production switches from an ‘all’ direct to an ‘all’ indirect mechanism, the latter situation being comparable to the decay of 125I free in solution. These experimental findings were correlated with theoretical expectations based on microdosimetry.

Amination of Ketones by Employing Two New (S)‐Selective ω‐Transaminases and the His‐Tagged ω‐TA from Vibrio fluvialis

AbstractTwo recently identified (S)‐selective ω‐transaminases (ω‐TAs) that originate from Paracoccus denitrificans (Strep‐PD‐ωTA, cloned with an N‐terminal Strep‐tag II) and Pseudomonas fluorescens (PF‐ωTA) were employed for the asymmetric amination of selected prochiral ketones. The substrates tested were transformed into optically pure amines (>99 % ee) with high conversion (up to >99 %). The ω‐TAs led to higher conversion in the absence of dimethyl sulfoxide as a cosolvent than in its presence (15 %, v/v). Additionally, it was shown that a His‐tagged recombinant transaminase from Vibrio fluvialis (His‐VF‐ωTA, cloned with an N‐terminal His6‐tag) showed for a single substrate, ethyl acetoacetate, significantly higher stereoselectivity for the amination compared to the corresponding commercial enzyme preparation (>99 vs. 50 %).

The HepaRG cell line is suitable for bioartificial liver application

For bioartificial liver application, cells should meet the following minimal requirements: ammonia elimination, drug metabolism and blood protein synthesis. Here we explore the suitability of HepaRG cells, a human cell line reported to differentiate into hepatocyte clusters and surrounding biliary epithelial-like cells at high density and after exposure to dimethyl sulfoxide (DMSO). The effect of carbamoyl-glutamate (CG), an activator of urea cycle enzyme carbamoylphosphate synthetase (CPS) was studied additionally. The effects of DMSO and/or CG were assessed in presence of 15NH 4Cl on HepaRG cells in monolayer. We tested hepatocyte-specific functions at transcript and biochemical level, cell damage parameters and performed immunostainings. Ureagenesis, ammonia/galactose elimination and albumin, glutamine synthetase and CPS transcript levels were higher in −DMSO than +DMSO cultures, probably due to a higher cell content and/or cluster-neighbouring regions contributing to their functionality. DMSO treatment increased cytochrome P450 ( CYP) transcript levels and CYP3A4 activity, but also cell damage and repressed hepatic functionality in cluster-neighbouring regions. The levels of ammonia elimination, apolipoprotein A-1 production, and transcription of CYP3A4, CYP2B6 and albumin reached those of primary hepatocytes in either the + or −DMSO cultures. Preconditioning with CG increased conversion of 15NH 4Cl into 15N-urea 4-fold only in −DMSO cultures. Hence, HepaRG cells show high metabolic and synthetic functionality in the absence of DMSO, however, their drug metabolism is only high in the presence of DMSO. An unparalleled broad hepatic functionality, suitable for bioartificial liver application, can be accomplished by combining CG treated −DMSO cultures with +DMSO cultures.

Antioxidant properties of dimethyl sulfoxide and its viability as a solvent in the evaluation of neuroprotective antioxidants

Introduction Dimethyl sulfoxide is an amphiphilic compound whose miscibility with water and its ability to dissolve lipophilic compounds make it an appreciated solvent in biomedical research. However, its reported antioxidant properties raise doubts about its use as a solvent in evaluating new antioxidants. The goal of this investigation was to evaluate its antioxidant properties and carry out a comparative study on the antioxidant properties of some known neuroprotective antioxidants in the presence and absence of dimethyl sulfoxide. Methods The antioxidant properties of dimethyl sulfoxide were studied in rat brain homogenates by determining its ability to reduce both lipid peroxidation (TBARS formation) and protein oxidation (increase in protein carbonyl content and decrease in free thiol content) induced by ferrous chloride/hydrogen peroxide. Its ability to reduce the production of hydroxyl radicals by 6-hydroxydopamine autoxidation was also estimated. The same study was also performed with three known antioxidants (α-phenyl- N- tert-butylnitrone; 2-methyl-2-nitrosopropane; 5,5-dimethyl-1-pyrroline N-oxide) in the presence and absence of dimethyl sulfoxide. Results Our results showed that dimethyl sulfoxide is able to reduce both lipid peroxidation and protein carbonyl formation induced by ferrous chloride/hydrogen peroxide in rat brain homogenates. It can also reduce the production of hydroxyl radicals during 6-hydroxydopamine autoxidation. However, it increases the oxidation of protein thiol groups caused by ferrous chloride/hydrogen peroxide in rat brain homogenate. Discussion Despite the here reported antioxidant and pro-oxidant properties of dimethyl sufoxide, the results obtained with α-phenyl- N- tert-butylnitrone, 2-methyl-2-nitrosopropane, and 5,5-dimethyl-1-pyrroline N-oxide corroborate the antioxidant properties attributed to these compounds and support the potential use of dimethyl sulfoxide as a solvent in the study of the antioxidant properties of lipophilic compounds. Conclusion Dimethyl sulfoxide is a very useful solvent that may be used at relatively low concentrations in the development of new antioxidants with neuroprotective properties.

Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells

Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells (ASCs) could increase the usefulness of these cells in tissue engineering and regenerative medicine. To this end, we investigated the post-freeze/thaw viability and apoptotic behavior of Passage 1 (P1) adult stem cells (ASCs) in 11 different media: (i) the traditional media containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO), (ii) DMEM with 80% human serum (HS) and 10% DMSO, (iii) DMEM with 1% methyl cellulose (MC) and 10% of either HS or FCS or DMSO, and (iv) DMEM with 0%, 2%, 4%, 6%, 8%, or 10% DMSO. Approximately 1 mL (10(6) cells/mL) of P1 ASCs were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees C water bath (1-2 min of agitation), resuspended in culture media, and seeded in separate wells of a 6-well plate for a 24-h incubation period at 37 degrees C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO (and the presence of MC) significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 2% DMSO and DMEM was comparable with that obtained in freezing media with 10% DMSO and 80% serum (HS or FCS), that is, approximately 84% +/- 5% and approximately 84% +/- 8%, respectively. Adipogenic and osteogenic differentiation behavior of the frozen thawed cells was also assessed using histochemical staining. Our results suggest that post-thaw ASC viability, adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum but with a minimal concentration of 2% DMSO in DMEM.

Investigation of Freezing- and Thawing-Induced Biological, Chemical, and Physical Changes to Enoxaparin Solution

This study investigated the effect of freezing and thawing on the biological, physical, and chemical properties of enoxaparin solution. Solutions were frozen and thawed under different conditions, in the presence or absence of dimethyl sulfoxide (DMSO) or 1,2-propanediol (1,2-PD), and the antifactor Xa (AFXa) activity was determined. Enoxaparin solution lost more than 60% of its AFXa activity when thawed rapidly after freezing at −196°C. The loss of AFXa activity was less with higher freezing temperatures and increased with the number of freeze/thaw cycles, but was independent of the duration of freezing. Slow freezing to −196°C with rapid thawing, or rapid freezing with slow thawing, resulted in negligible loss of AFXa activity. The loss of AFXa activity did not involve the loss of N-sulfate groups, the breakdown of glycosidic bonds or the glassy state transition. Controlling the freezing or thawing conditions, dilution with water or addition of a small percentage of DMSO ameliorated the loss of enoxaparin AFXa activity. The loss in AFXa activity was found by size exclusion chromatography to be primarily due to aggregation and was reversed by sonication in the presence of DMSO. These results may provide insight into solutions for the long-term storage of concentrated or diluted enoxaparin.

Contributions of Direct and Indirect Actions in Cell Killing by High-LET Radiations

The biological effects of radiation originate principally in damages to DNA. DNA damages by X rays as well as heavy ions are induced by a combination of direct and indirect actions. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action without affecting direct action. Exponentially growing Chinese hamster V79 cells were exposed to high-LET radiations of 20 to 2106 keV/mum in the presence or absence of DMSO and their survival was determined using a colony formation assay. The contribution of indirect action to cell killing decreased with increasing LET. However, the contribution did not reach zero even at very high LETs and was estimated to be 32% at an LET of 2106 keV/mum. Therefore, even though the radiochemically estimated G value of OH radicals was nearly zero at an LET of 1000 keV/mum, indirect action by OH radicals contributed to a substantial fraction of the biological effects of high-LET radiations. The RBE determined at a survival level of 10% increased with LET, reaching a maximum value of 2.88 at 200 keV/mum, and decreased thereafter. When the RBE was estimated separately for direct action (RBE(D)) and indirect action (RBE(I)); both exhibited an LET dependence similar to that of the RBE, peaking at 200 keV/mum. However, the peak value was much higher for RBE(D) (5.99) than RBE(I) (1.89). Thus direct action contributes more to the high RBE of high-LET radiations than indirect action does.

Pathogenic role of natural killer T and natural killer cells in acetaminophen-induced liver injury in mice is dependent on the presence of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) is commonly used in biological studies to dissolve drugs and enzyme inhibitors with low solubility. Although DMSO is generally thought of as being relatively inert, it can induce biological effects that are often overlooked. An example that highlights this potential problem is found in a recent report demonstrating a pathogenic role for natural killer T (NKT) and natural killer (NK) cells in acetaminophen-induced liver injury (AILI) in C57Bl/6 mice in which DMSO was used to facilitate acetaminophen (APAP) dissolution. We report that NKT and NK cells do not play a pathologic role in AILI in C57Bl/6 mice in the absence of DMSO. Although AILI was significantly attenuated in mice depleted of NKT and NK cells prior to APAP treatment in the presence of DMSO, no such effect was observed when APAP was dissolved in saline. Because of this unexpected finding, the effects of DMSO on hepatic NKT and NK cells were subsequently investigated. When given alone, DMSO activated hepatic NKT and NK cells in vivo as evidenced by increased NKT cell numbers and higher intracellular levels of the cytotoxic effector molecules interferon-gamma (IFN-gamma) and granzyme B in both cell types. Similarly, when used as a solvent for APAP, DMSO again increased NKT cell numbers and induced IFN-gamma and granzyme B expression in both cell types. These data demonstrate a previously unappreciated effect of DMSO on hepatic NKT and NK cells, suggesting that DMSO should be used cautiously in experiments involving these cells.

Synthesis and Electrochemical Behavior of Single-Crystal Magnetite Nanoparticles

Water-dispersed magnetite nanoparticle synthesis from iron(II) chloride in dimethyl sulfoxide (DMSO)−water solution at different DMSO−water ratios in alkaline medium was reported. TEM and XRD results suggest a single-crystal formation with mean particle size in the range 4−27 nm. Magnetic nanoparticles are formed by the oxidative hydrolysis reaction from green rust species that leads to FeOOH formation, followed by autocatalysis of the adsorbed available Fe(II) on the FeOOH surfaces. The available hydroxyl groups seem to be dependent on the DMSO−water ratio due to strong molecular interactions presented by the solvent mixture. Goethite phase on the magnetite surface was observed by XRD data only for sample synthesized in the absence of DMSO. In addition, cyclic voltammetry with carbon paste electroactive electrode (CV-CPEE) results reveal two reduction peaks near 0 and +400 mV associated with the presence of iron(III) in different chemical environments related to the surface composition of magnetite nanoparticles. The peak near +400 mV is related to a passivate thin layer surface such as goethite on the magnetite nanoparticle, assigned to the intensive hydrolysis reaction due to strong interactions between DMSO−water molecules in the initial solvent mixture that result in a hydroxyl group excess in the medium. Pure magnetite phase was only observed in the samples prepared at 30% (30W) and 80% (80W) water in DMSO in agreement with the structured molecular solvent cluster formation. The goethite phase present on the magnetite nanoparticle surface like a thin passivate layer only was detectable using CV-CPEE, which is a very efficient, cheap, and powerful tool for surface characterization, and it is able to determine the passivate oxyhydroxide or oxide thin layer presence on the nanoparticle surface.

Existence of a New Reactive Intermediate Oxygen Species in Hypoxanthine and Xanthine Oxidase Reaction

We investigated a hypoxanthine (HPX) and xanthine oxidase (XOD) reaction by using a luminol analog 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-mediated chemiluminescence (CL) response. Addition of a high activity of superoxide dismutase (SOD), a potent O2* scavenger, and of a high concentration of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a potent spin trapping agent, diminished completely the CL response. Whereas a high concentration of dimethyl sulfoxide (DMSO), as a potent *OH scavenger could not attain to the complete diminishment of the CL response. It has been reported that luminol monoanion reacts with *OH to form luminol radical, and then resultant luminol radical reacts with O2* to elicit CL response. Complete scavenging for *OH is assumed to result in lack of luminol radical, which in turn induces lack of CL response. However, our results did not support the idea. Furthermore, we examined the effect of L-012 on the DMPO-OOH formation in the presence or absence of DMSO in the HPX-XOD system by applying an electron spin resonance (ESR)-spin trapping method. The DMPO-OOH formation was inhibited even in the presence of DMSO, and the rate constant (k2) between L-012 and O2* obtained in the presence of DMSO was 9.77 x 10(2) M(-1) s(-1) and the constant in the absence of DMSO was 2.97 x 10(3) M(-1) s(-1). The data suggests that L-012 is converted to a radical form that reacts with O2* even under the conditions of the absence of *OH. From these, we postulate that the existence of a reactive intermediate oxygen species in the HPX-XOD system.