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Organic solvent systems for 31P nuclear magnetic resonance analysis of lecithin phospholipids: applications to two-dimensional gradient-enhanced 1H-detected heteronuclear multiple quantum coherence experiments.

31P NMR of lipid extracts is a reproducible, rapid, and nondegradative method for qualitative and quantitative analyses of phospholipid mixtures. This analysis, however, is hampered by the instability of the solvent system commonly used for NMR spectroscopy (CHCl3/ CH3OH/H2O-EDTA). In this work we have investigated the effects of several monophasic solvent mixtures to overcome this disadvantage. Among these mixtures we have selected a solution of triethylamine, dimethylformamide, and guanidinium chloride (Et3N/DMF-GH+) as the most efficient system. In this solvent the chemical shift dispersion of the 31P signals is about four times the frequency range observed in the standard chloroform-methanol-water system. Moreover, the stability of this solvent, as a monophasic system, allows easy reproducibility of the analysis. The use of two-dimensional 1H-31P gradient-enhanced heteronuclear multiple quantum coherence experiments can further exploit the higher resolution of the signals obtained with this solvent system for the structure elucidation of known and unidentified phospholipids.

Acetamides↗

Dimethyl sulfoxide and related polar compounds enhance infection of human T cells with HIV-1 in vitro.

We observed the enhancing effect of dimethylsulfoxide (DMSO) on infection of human T cells with human immunodeficiency virus type 1 (HIV-1). Similar enhancing effects were also found in related polar chemicals such as dimethylformamide, hexamethylenebisacetamide, sodium butyrate and retinoic acid. In acute infection of the human MT-4 T cell line, DMSO at a concentration of 1% reduced the amounts of HIV-1 required to establish similar infection by one log. Furthermore, infection of peripheral blood lymphocytes with HIV-1 was also augmented several times by DMSO. HIV-1 production from persistently infected human T cell lines, but not monocytic cell lines, was enhanced by DMSO and related polar chemicals. DMSO enhanced transcription of HIV-1 RNA in persistently infected T cell lines, and the enhancing effect of DMSO on HIV-1 production was inhibited by staurosporine, a protein kinase inhibitor. These findings suggested that DMSO enhanced HIV-1 infection of T cells mainly at the step of transcription of viral RNA.

Acetamides↗

Glass-forming tendency in the system water-dimethyl sulfoxide.

The glass-forming tendency on cooling and the stability of the wholly amorphous state on warming have been previously reported for many cryoprotective solutions. However, unlike the other solutions, those of dimethyl sulfoxide (Me(2)SO) have not been studied on cooling. In this paper, the glass-forming tendency of Me(2)SO aqueous solutions has been measured for solutions containing 40, 43, 45, and 47.5% (w/w) Me(2)SO. At a concentration of 45% (w/w), the glass-forming tendency decreases in the following order: levo-2, 3-butanediol, 1,3-butanediol, 1,2-propanediol, 1,2,3-butanetriol, dimethyl sulfoxide, dimethylformamide, diethylformamide, 1, 4-butanediol, ethylene glycol, glycerol, 1,3-propanediol. New measurements have also been made on warming the Me(2)SO solutions.

Butylene Glycols↗

Estrogen induction of plasma vitellogenin in the Kemp's ridley sea turtle (Lepidochelys kempi).

Estrogen stimulation of the production of teh yolk protein precursor vitellogenin was demonstrated in immature Kemp's ridley sea turtles. 17beta-Estradiol injection elicited an increase in serum estrogen, protein, protein phosphorus, and total calcium within 7 days. Associated with these changes was the appearance of a single, dimethylformamide-precipitable, 205-kDa estradiol-induced serum protein, which became the predominant serum protein identified by polyacrylamide gel electrophoresis. The effects of estradiol injection were maintained for 3 months following termination of estradiol administration, despite a decline in serum estrogen levels. Although studies in other species have indicated that changes in circulating vitellogenin levels can influence thyroid hormone transport, no changes were observed throughout the study in total blood levels of thyroxine or triiodothyronine or protein binding of these hormones. We conclude that in the immature Kemp's ridley estrogen induces the synthesis and secretion of a vitellogenin which resembles that of other reptiles, but which does not influence thyroid hormone transport. The prolonged presence of vitellogenin in the blood may be due to a lack of an active ovarian uptake mechanism in these immature animals.

Animals↗

Procoagulant and fibrinolytic properties of bovine endothelial cells treated with monocrotaline pyrrole.

Administration to rats of monocrotaline pyrrole (MCTP), a putative metabolite of the pyrrolizidine alkaloid, monocrotaline, causes pulmonary microvascular thrombosis that is associated with vascular remodeling and pulmonary hypertension. It is possible that vascular thrombi contribute to the lesions that occur after MCTP treatment. Since MCTP is toxic to pulmonary endothelial cells and because the pulmonary endothelium is in a unique position to influence procoagulant and fibrinolytic reactions in the lungs, we examined changes in the procoagulant and fibrinolytic properties of cultured pulmonary artery endothelial cells exposed to MCTP to see if they might favor thrombosis. Monolayers of confluent bovine pulmonary arterial endothelial cells received a single administration of MCTP or N,N-dimethylformamide vehicle, and the media or lysates were examined at 4, 24, 48, or 120 hr after treatment. MCTP caused a time-dependent cell detachment and an increase in release of lactate dehydrogenase into culture medium. Although cell numbers decreased dramatically with time, the protein concentration of cell monolayer lysates was unchanged by treatment. MCTP treatment caused no change in the amount of tissue factor activity/micrograms cellular protein in bovine endothelial cell lysates and only a small increase in the activity of Factor V in the culture medium at 120 hr. In addition, the medium from bovine endothelial cells treated with MCTP had a time-dependent increase in the activity of plasminogen activators and a decrease in the activity of plasminogen activator inhibitors. Thus bovine endothelial cells exposed to MCTP in vitro do not produce changes in these procoagulant or fibrinolytic properties that would explain the thrombosis observed in vivo.

Animals↗

An analysis of dimethylsulfoxide-induced action potential block: a comparative study of DMSO and other aliphatic water soluble solutes.

A series of water soluble aliphatic solutes were chosen for study. Fifty percent effective doses (ED50) to block propagated compound action potentials (AP's) were obtained by examining dose-response relations for each solute. All solutes used were liquids at room temperature and are typically used as solvents. The solutes studied were dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, acetone, and hexamethylphosphoramide (HMPA); the octanol/water partition coefficients for these test substances form an ordered sequence that increased 40-fold from DMSO to HMPA. AP's were recorded from desheathed frog sciatic nerves using the sucrose-gap technique; test solutes were added to Ringer's solution and applied externally to the nerve. ED50's for the solutes could be predicted as a function of the molar volume (dV/dn), polarity (P), and the hydrogen bond acceptor basicity (beta). Voltage-clamp experiments employing the vaseline-gap technique on single muscle fibers showed that each solute reduced Na+ current with little change in their kinetics at all voltages studied. Experiments using DMSO or DMF showed that Na+ channel block alone is insufficient to explain the respective ED50 values of AP block. Experiments conducted using a chloride transport-sensitive membrane fluidity assay, using rat pancreas secretory granules, suggested that each of the solutes act to increase membrane fluidity at doses below and above ED50 values. Light microscopic observations of fixed thick sections of whole nerves previously exposed to DMSO or DMF show structural changes; however, ED50 values cannot be simply explained by osmotic alterations of nerve structure. ED50's are likely to be produced by a combination of effects including osmotically induced nerve structural changes, ion channel block, and fluidity changes. The toxicity (lethal doses or toxic concentrations) of each of these five solutes correlates well with the ED50 and could be predicted as a function of dV/dn, P, and beta.

Acetamides↗

A morphological and experimental study of gonadal sex differentiation in the rainbow trout, Salmo gairdneri.

Gonadal sex differentiation in rainbow trout takes place between day (D) 45 and D 55 after fertilization. Until D 400 the male GSI is maintained at about 0.4%, and that of the female at about 1%. Treatment with N,N-dimethylformamide (DMF) influences the sex ratio and GSI, and is therefore unsuitable as a solvent for steroids to be added to the aquarium water for examination of their effects on gonadal sex differentiation. Triton X-100 has no such effect, and is used as the steroid solvent in the present experiments. Progesterone (300 microgram/l) administered for 4 weeks from hatching, or from D 43, significantly affects the sex ratio in favour of females. Methyltestosterone given from hatching results in sterilization of the gonads, especially when the treatment is carried out for 8 weeks, or when a relatively high dose (300 microgram/l) is used for 4 weeks. When given from D 43 methyltestosterone has a masculinizing effect. It is concluded that progesterone and androgen(s) are important in inducing the onset of ovarian and testis development, respectively.

Animals↗

Proton-induced dismutation of superoxide in aprotic media by bile pigments.

Cyclic voltammetry of molecular oxygen in aprotic media (dimethylformamide) and in the presence of bilirubin and other bile pigments shows that superoxide anion (O2-.) undergoes proton-induced dismutation. Lactam hydrogens of bile pigments are sufficiently acid to induce (O2-.) disproportionation to O2 and H2O2. Because of its characteristic lipophilic behavior, a biological role for natural bilirubin similar to that of other non-enzymatic lipophilic scavengers of (O2-.) is suggested.

Bile Pigments↗

Preclinical evaluation of biotin labeling for red cell survival testing.

Biotin labeling of red cells was tested in dogs as a preclinical study for cell survival. Red cells were labeled with either spacered Biotin-X-NHS (BxNHS) or water-soluble biotin compounds. After reinfusion, biotinylated red cells were detected in small blood samples (5 microliters) with flow cytometry. Improved BxNHS labeling allows an easy detection of positive red cells for almost 100 days, whereas labeling with watersoluble compounds-despite strong labeling during the first days-results in a decrease of label, which prevented a discrimation between labeled and negative cells after about 4 weeks. When biotin labeling of red cells was compared with 51Cr labeling, slopes of red cell survival were quite similar after the latter were corrected for elution. Survival slopes were linear, and the mean survival time was t = 93 d. In two blood-donor dogs the slopes of red cell survival where log linear and the mean survival time was t = 45 d. In conclusion, BxNHS, but not the water-soluble biotin compounds, is a good nonradioactive, nontoxic alternative for red cell survival studies. No health hazards are to be expected from the very low dose of Dimethylformamide, which is used as a solvent for biotin-x-NHS.

Animals↗

Exposure to solvents in a synthetic leather manufacturing plant.

The objective of this study was to evaluate the exposure of synthetic leather workers to dimethylformamide (DMF), epichlorohydrin (ECH) and toluene, in a manufacturing plant. The correlation between biological and environmental monitoring for DMF was also investigated. Environmental monitoring of the three solvents included personal and area sampling based on time of day (morning and afternoon). Urine samples were taken at the end of the shift and urinary N-methylformamide (NMF) was then used to biologically monitor DMF levels. Results for solvent concentrations based on air sampling were considerably higher in area than in personal sampling. Of 41 area samples, 15 (36.6%) and five (12.2%) exceeded permissible exposure levels for DMF and ECH, respectively, compared with 22 (28.6%) and three (3.9%) based on 77 personal samples. Overall, urinary NMF concentrations were lower than the Biological Exposure Index (BEI) suggested by the American Conference of Governmental Industrial Hygienists (ACGIH). A significant correlation (r = 0.32; P < 0.05) was found between environmental and biological monitoring. Even though urinary NMF concentration was within permissible levels for Taiwan, the authors recommend that immediate measures be taken to decrease DMF and ECH concentrations in synthetic leather manufacturing plants.

Adult↗

Titanium(IV) targets phosphoesters on nucleotides: implications for the mechanism of action of the anticancer drug titanocene dichloride.

Abstract Reactions between the anticancer drug titanocene dichloride (Cp2TiCl2) and various nucleotides and their constituents in aqueous solution or N,N-dimethylformamide (DMF) have been investigated by 1H and 31P NMR spectroscopy and in the solid state by IR spectroscopy. In aqueous solution over the pH* (pH meter reading in D2O) range 2.3-6.5, CMP forms one new species with Ti(IV) bound only to the phosphate group. In acidic media at pH*<4.6, three species containing titanocene bound to the phosphate group of dGMP, AMP, dTMP and UMP are formed rapidly. The bases also appear to influence titanocene binding. Only one of these Ti(IV)-bound species can be detected in the pH* range of 4.6-6.5 in each case. The order of reactivity towards Cp2TiCl2(aq) at pH* ca. 3 is GMP>TMP approximately AMP > CMP. At pH* > 7.0, hydrolysis of Cp2TiCl2 predominated and little reaction with the nucleotides was observed. Binding of deoxyribose 5'-phosphate and 4-nitrophenyl phosphate to Cp2TiCl2(aq) via their phosphate groups was detected by 31P NMR spectroscopy, but no reaction between Cp2TiCl2(aq) and deoxyguanosine, 9-ethylguanine or deoxy-D-ribose was observed in aqueous solution. The nucleoside phosphodiesters 3',5'-cyclic GMP and 2',3'-cyclic CMP did not react with Cp2TiCl2(aq) in aqueous solution; however, in the less polar solvent DMF, 3',5'-cyclic GMP coordination to [Cp2Ti]2+ via its phosphodiester group was readily observed. Binding of titanocene to the phosphodiester group of the dinucleotide GpC was also observed in DMF by 31P NMR. The nucleoside triphosphates ATP and GTP reacted more extensively with Cp2TiCl2(aq) than their monophosphates; complexes with bound phosphate groups were formed in acidic media and to a lesser extent at neutral pH. Cleavage of phosphate bonds in ATP (and GTP) by Cp2TiCl2(aq) to form inorganic phosphate, AMP (or GMP) and ADP (or GDP) was observed in aqueous solutions. In addition, titanocene binding to ATP was not inhibited by Mg(II), but the ternary complex titanocene-ATP-Mg appeared to form. These reactions contrast markedly with those of the drug cisplatin, which binds predominantly to the base nitrogen atoms of nucleotides and only weakly to the phosphate groups. The high affinity of Ti(IV) for phosphate groups may be important for its biological activity.

Adenosine Monophosphate↗

Influence of water miscible organic solvents on alpha-chymotrypsin in solution and immobilized on Eupergit CM.

The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N'-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of alpha-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20 degrees C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.

Acetonitriles↗

Coupling of peptides to protein carriers by mixed anhydride procedure.

Carboxyl groups of succinylated bovine serum albumin were activated by isobutylchloroformate in dimethylformamide solution. Subsequent reaction of the mixed anhydride with amino groups of the added peptide provided rapid and efficient coupling of peptide to protein. For different peptides the yield of coupling was equal to 40-100%. These values corresponded to 20-50 mol peptide bound/mol protein. Immunization of rabbits with these conjugates produced antisera to peptides with titers of 1:1000-1:3000 (estimated by enzyme-linked immunosorbent assay).

Albumins↗

Differential effects of denaturing agents on acetylcholinesterase: insensitivity of the reaction of methanesulfonyl fluoride compared to diisopropylphosphorofluoridate and p-nitrophenyl acetate.

The effects of various agents that could be expected to perturb enzyme structure in a non-specific and reversible manner (alcohols, dimethylsulfoxide, dimethylformamide, dinitrobenzene, urea and guanidine - HCl) have been determined on reaction of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with a substrate, p-nitrophenyl acetate, and two irreversible inhibitors, diisopropylphosphorofluoridate and methanesulfonyl fluoride. In all three of these reactions an acyl group (acetyl, phosphoryl or sulfonyl respectively) bonds covalently with the active center of the enzyme. As expected, the reactions of p-nitrophenyl acetate and diisopropylphosphorofluoridate were severely retarded by most of these agents. By contrast, reaction of methanesulfonyl fluoride was usually depressed to a far smaller degree, and in two cases was faster. These findings are of interest in connection with: (1) differing requirements for the integrity of tha active center in catalysis with various substrate analogs, and (2) the mechanism by which cationic substrate analogs accelerate reaction of the enzyme with methanesulfonyl fluoride.

Acetylcholinesterase↗

High yield coupling of peptides to protein carriers.

Coupling of peptides to protein carriers has been achieved at a high level of peptide incorporation. Bovine serum albumin was used as the model carrier protein and several peptides were tested in the coupling reaction. The peptides were coupled to succinylated bovine serum albumin after conversion of its carboxyl side chains to the reactive p-nitrophenyl ester group by reaction with p-nitrophenol/N,N'-dicyclohexylcarbodiimide in anhydrous dimethylformamide. The reaction afforded succinyl-albumin-peptide conjugates that had high level of peptide incorporation (18-35 mol peptide/mol albumin). In addition to the high level of peptide coupling, the reaction avoids the production of polymeric forms of peptide, protein or conjugate.

Carrier Proteins↗

Suicide inhibition as a likely cause of variable specific activity in trimethylamine dehydrogenase from bacterium W3A1.

Trimethylamine hydrogenase isolated from bacterium W3A1 grown on dimethylamine was of variable, but low specific activity and had modified spectral properties. Chemical analyses for Fe, S and P indicated that the [4Fe-4S] clusters of the modified enzyme are intact and that the covalently bound flavin is probably present, but in modified form. A peptide with absorbance maximum at 358 nm and fluorescence excitation and emission maxima in dimethylformamide at 358 nm and 495 nm, respectively, was isolated by gel chromatography and HPLC of tryptic peptides of acetamidylated, modified trimethylamine dehydrogenase. These spectral properties are similar to those of 4a- or 5a-substituted flavins and suggest that the enzyme had been modified by in vivo reaction with a suicide inhibitor. This inhibitor, or a compound giving rise to it, seems to be present in a commercial source of dimethylamine.

Bacteria↗

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts.

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.

Animals↗