Dimethylformamide Preserves the Integrity of Cryopreserved Goat Semen in a Soybean Lecithin-Based Extender.

This study investigated the cryoprotectant effects of dimethylformamide (DMF), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) as substitutes for glycerol (GLY) in a soybean lecithin (SL)-based extender in the cryopreservation of buck sperm. In this study, the semen of three Saanen bucks was individually extended in SL supplemented with 5% GLY (control), DMF, EG, or DMSO. After this, the extended semen was cryopreserved and two straws from each group were thawed (37°C for 30 seconds), pooled, and analyzed for sperm motion parameters, plasma membrane integrity (PMI), acrosomal integrity (ACI), and high mitochondrial membrane potential (HMMP). Samples were analyzed after 15 minutes (T0) and after 2 hours of incubation at 37°C (T2). The results revealed higher values of motility (total and progressive) and sperm motion parameters for DMF than the other cryoprotectants (p < 0.0001). PMI and HMMP did not differ (p > 0.05) between GLY and DMF, but ACI was higher (p < 0.01) for DMF compared with GLY. Based on these results, DMF and GLY samples were used in heterologous in vitro fertilization assays by using bovine oocytes (n = 337) obtained from a slaughterhouse. No differences (p > 0.05) were observed between GLY and DMF for unfertilized (GLY: 38.8%; DMF: 25.33%), pronucleus (GLY: 25.68%; DMF: 27.92%), and cleavage rates (GLY: 35.52%; DMF: 46.75%). Based on these results, it is concluded that DMF preserves sperm motion characteristics and ACI better than GLY, EG, and DMSO, and it is the penetrating cryoprotectant of choice for the cryopreservation of buck sperm in SL extender.

Sulfated vizantin induces formation of macrophage extracellular traps.

Vizantin is an insoluble adjuvant that activates macrophages and lymphocytes. Recently, 2,2',3,3',4,4'-hexasulfated-vizantin (sulfated vizantin), which enables solubilization of vizantin, was developed by the present team. Sulfated vizantin was found to enhance bactericidal activity against multi-drug resistant Pseudomonas aeruginosa in RAW264.7 cells. In addition, spread of P. aeruginosa was inhibited in RAW264.7 cells treated with sulfated vizantin. When only sulfated vizantin and P. aeruginosa were incubated, sulfated vizantin did not affect growth of P. aeruginosa. Formation of DNA-based extracellular traps (ETs), a novel defense mechanism in several types of innate immune cells, helps to eliminate pathogens. In the present study, ET-forming macrophages constituted the majority of immune cells. Sulfated vizantin induced ET formation in RAW264.7 cells, whereas a Ca-chelating reagent, EDTA, and T-type calcium channel blocker, tetrandrine, inhibited ET formation and attenuated inhibition of spread of P. aeruginosa in sulfated vizantin-treated cells. Thus, sulfated vizantin induces ET formation in phagocytic cells in a Ca-dependent manner, thus preventing spread of P. aeruginosa. Hence, sulfated vizantin may be useful in the management of infectious diseases.

Predicting changes in aquatic toxicity of chemicals resulting from solvent or dispersant use as vehicle.

The influence of two vehicles (N,N-dimethylformamide [DMF] as solvent and polyoxyethylene hydrogenated castor oil [HCO-40] as a dispersant) on the acute toxicity of eight hydrophobic chemicals with a non-specific mode of action to Daphnia magna was investigated according to the OECD Guidelines for the Testing of Chemicals, No. 202. An increased 48-h EC50 value for D. magna or reduced toxicity resulting from the addition of HCO-40 to the test medium was observed for five of the eight chemicals examined. Each of eight chemicals was dissolved in water at a concentration of either 10 mg/L or 1.0 mg/L, with or without DMF or HCO-40. Silicone film as a model of a biological membrane was then immersed in each solution, and the concentration of each chemical in the water was monitored until equilibrium was reached for each test substance, after which the adsorbed amount of each chemical was determined. The amounts of p-pentylphenol and four other substances with log Pow (1-octanol/water partition coefficient) values greater than 3.4 adsorbed onto the silicone film decreased with increasing concentrations of HCO-40. However, 3-chloro-4-fluoronitrobenzene and two other substances with log Pow values less than 2.6 demonstrated no changes in adsorption with either increasing HCO-40 concentration or the addition of DMF. The reduced adsorption in the presence of a vehicle on the silicone film correlated closely with changes in toxicity. These results indicate that the methodology developed in this study enables the prediction of changes in toxicity resulting from the addition of vehicles to a test system.

THE COMBINED USE OF HONEY, GARLIC (ALLIUM SATIVUM L.) AND SKIMMED MILK AS AN EXTENDER FOR CHILLING SHEEP SEMEN.

BACKGROUND: Sugars are the energetic source for sperm to maintain the metabolic process, and the antibiotics slow down sperm degradation. OBJECTIVE: To study the effects of rosemary honey as energy source and cryoprotectant in combination with garlic as a natural antibiotic on the quality of ram spermatozoa upon cooling. MATERIALS AND METHODS: The ejaculates from three rams were evaluated at different times during cooling to determine its post-dilution quality. RESULTS: Glycerol and dimethylformamide in conjunction with honey and garlic significantly improve the survival of spermatozoa. CONCLUSION: The addition of honey and garlic reduces sperm deterioration when stored at 4 degree C.

Dimethylformamide as a cryoprotectant for canine semen diluted and frozen in ACP-106C.

The objective was to assess the effect of adding various concentrations of dimethylformamide on characteristics of canine semen diluted in powdered coconut water (ACP-106C; ACP Biotecnologia, Fortaleza, CE, Brazil) and frozen at -196°C. Fifteen ejaculates were collected by manual stimulation from five adult Boxer dogs. The sperm-rich fraction was diluted in ACP-106C (ACP Biotecnologia) containing 10% egg yolk and divided into four aliquots. The cryoprotectants used for each aliquot were 6% glycerol (control group; CG) or 2%, 4%, or 6% dimethylformamide (DF2, DF4, and DF6, respectively). After thawing, total motility (mean ± SEM) for CG (58.4 ± 24.6) was higher (P < 0.05) than that of the other groups (2% dimethylformamide, 24.4 ± 12.3; 4% dimethylformamide, 26.5 ± 16.1; and 6% dimethylformamide, 21.7 ± 17.9). Furthermore, there was a greater percentage of fast, average, and slow moving sperm (assessed with computer-aided semen analysis; CASA) in CG in comparison with the other three groups. Therefore, based on concentrations tested in this study, dimethylformamide, together with ACP-106C (ACP Biotecnologia) and 10% egg yolk as a diluent, yielded unsatisfactory in vitro results for freezing canine semen.

Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: an in vitro study.

AIM: To determine if Tulsi (Ocimum sanctum) extract has an antimicrobial activity against Streptococcus mutans and to determine which concentration of Tulsi (Ocimum sanctum) extract among the 15 concentrations investigated has the maximum antimicrobial activity. SETTING AND DESIGN: Experimental design, in vitro study, Lab setting. MATERIALS AND METHODS: Ethanolic extract of Tulsi was prepared by the cold extraction method. The extract was then diluted with an inert solvent, dimethyl formamide, to obtain 15 different concentrations (0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7% 8%, 9%, 10%) of the extract. 0.2% chlorhexidine was used as a positive control and dimethyl formamide was used as a negative control. The extract, along with the controls, was then subjected to microbiological investigation to determine which concentration among the 15 different concentrations of the extract gave a wider inhibition zone against Streptococcus mutans. The zones of inhibition were measured in millimeters using a vernier caliper. RESULTS: At the 4% concentration of Tulsi extract, a zone of inhibition of 22 mm was obtained. This was the widest zone of inhibition observed among all the 15 different concentrations of Tulsi that were investigated. CONCLUSION: Tulsi extract demonstrated an antimicrobial property against Streptococcus mutans.

Method to screen substrates of apical sodium-dependent bile acid transporter.

Human apical sodium-dependent bile acid transporter (hASBT) is a potential prodrug target under study. Development of prodrugs that target hASBT may yield compounds with low solubility and/or susceptibility to hydrolysis. A transport uptake method is needed that increases compound solubility and avoids NaOH for cell lysis during postexperimental cell sample preparation. The overall goal was to develop an assay method to screen for hASBT uptake of novel compounds. The first objective was to determine the maximum cosolvent concentrations that are compatible with an hASBT active transport assay. The second objective was to develop a NaOH-free cell lysis method to process cell samples from these uptake studies. The following cosolvents were studied: dimethylacetamide (DMAC), dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, methanol, polyethylene glycol-400, propylene glycol, and dioxane. Initial studies included taurocholate flux studies across hASBT-Madin-Darby canine kidney monolayers using up to 10% cosolvent, as well as cytotoxicity studies. The effect of selected cosolvent concentrations on the hASBT Michaelis-Menten kinetic parameters was evaluated. Additionally, two acetonitrile-based cell lysis methods that do not use NaOH were evaluated in terms of percent sample recovery and hASBT kinetic parameters. Results showed that the maximum permissible cosolvent concentrations for hASBT uptake studies, without compromising assay results or causing cytotoxicity, are 1% DMAC, 1% DMF, 2.5% DMSO, 2.5% methanol, and 2.5% ethanol. Additionally, both NaOH-free, acetonitrile-based cell lysis methods provided similar recovery and hASBT results, compared to NaOH method. Hence, an assay method was developed to screen for active transport, allowing for cosolvents that can solubilize compounds and avoid NaOH sample treatment, which can otherwise degrade compound.

Effects of organic solvents on immobilized lipase in pectin microspheres.

Lipase from Brevibacillus agri 52 was found stable up to 90% diethylenglycol (DEG), glycerol (GLY), and 1,2 propanediol (1,2 PRO) at 37 degrees C for 1 h and the stability was reduced only approximately 20% after 12 h incubation, but in 40% dimethylsulfoxide (DMSO), lipase activity was stable only for 1 h. Inhibition of the biocatalysts with dimethylformamide (DMF) was detected at 20% solvent concentration. In water immiscible systems, the stability of lipase in n-hexane, n-tetradecane and n-heptane resembles the water activity, but in the presence of isobutanol, 1-hexanol, and butylbutirate, the stability was significantly reduced. Lipase 52 precipitates in the presence of 50% acetone or ethanol/water mixtures, but enzymatic activity was partially recovered by adding 20% GLY, DEG, 1,2 PRO, or DMSO to the reaction mixture. Furthermore, by increasing DEG in 70% DMF/DEG mixtures, the lipase activity was protected. Encapsulation of lipase in pectin gels cross-linked with calcium ions brings three to four times more enzymatic activity in 70% water miscible organic solvents compared to aqueous systems.

Optimization of nb-4 and hl-60 differentiation for use in opsonophagocytosis assays.

Production of effective vaccine formulations is dependent on the availability of assays for the measurement of protective immune responses. The development and standardization of in vitro human cell-based assays for functional opsonophagocytic antibodies require critical evaluation and optimization of the preparation of cells for the assay. We report evaluation of a number of protocols with two continuous cell lines (NB-4 and HL-60) for the provision of differentiated cells for use in functional assays. Flow cytometric analysis of CD11b antigen expression, as a marker of differentiation, indicated that all-trans-retinoic acid (ATRA) gave improved differentiation (>80% of cells differentiated at 96 h) when compared with dimethylformamide (DMF) (<60% of cells differentiated at 96 h). Morphological changes during differentiation toward a neutrophil-like phenotype were assessed by scanning electron microscopy. HL-60 and NB-4 cells treated with ATRA showed more spreading and flattening than cells treated with DMF, further evidence that they may have achieved a more differentiated phenotype. The number of cell divisions in culture appeared to be critical because cell lines maintained in exponential growth for >40 passages failed to express CD11b antigen or show morphological changes associated with differentiation after exposure to either differentiation-inducing reagent. Late-passage cells also demonstrated increased tolerance to DMF. Our results indicated that ATRA supplemented with vitamin D(3) and granulocyte colony-stimulating factor affords robust, rapid, and reproducible differentiation of both cell types.

Role of tumor necrosis factor alpha in hyperthermia-induced apoptosis of human leukemia cells.

We used the human myelomonoblastic leukemia cell line PLB-985 to study the effects of temperatures ranging from 37 degrees C to 43 degrees C for 1 h on the induction of apoptosis and cell cycle distribution in leukemia cells. The threshold temperature for the onset of apoptosis was 42 degrees C. Whereas hyperthermia exerted no effect on the expression of Bcl-2 and Bax, heat induced a >30-fold increase of tumor necrosis factor (TNF) alpha mRNA expression and a significant increase in TNF-alpha protein secretion. This endogenous production of TNF-alpha correlated directly with the temperature-induced apoptode effect. Blocking TNF-alpha expression via treatment with pyrrolidinedithiocarbamate or blocking TNF-alpha activity with neutralizing antibodies abrogated heat-provoked apoptosis. In addition, exposure of cell culture supernatant of heat-treated PLB-985 cells to untreated cells induced an apoptotic effect. These data indicate a TNF-a-mediated self eradication of the leukemia cells after heat exposure. Inducing apoptosis with wild-type TNF-alpha or p55 and p75 protein muteins demonstrated that this effect was mediated by the p55 receptor. Interestingly, the autocrine suicidal loop found in immature leukemia cells was lost after granulocytic differentiation with 0.5% N,N-dimethylformamide. These data should be of critical importance for the understanding of the biological impact of fever as well as for developing therapeutic approaches to malignant diseases

[Induction of quinone reductase and glutathion-s-transferase by dimethyl fumarate in rats].

OBJECTIVE: To assess induction of quinone reductase (QR) and glutathion-s-transferases (GSTs) by dimethyl fumarate (DMF) in major viscera of rats. METHODS: Fodder fed to male Wistar rats was supplemented with 2.0% DMF, and blood specimens were collected form the rats 6 h, 24 h, 72 h, 1 week and 2 weeks after feeding, respectively, and then animals were killed. QR and GSTs activities were determined in rats' gland stomach, liver, lung, kidney and heart, and also QR, LDH and GPT activities in their sera were measured with enzyme dynamic methods. RESULTS: Administration of DMF for 24 h, 72 h, 1 week and 2 weeks in rats can significantly increase activities of QR and GSTs in their gland stomach, liver, lung and kidney, as compared with those in controls (P < 0.0001, or P < 0.01), and their serum QR also significantly increased (P < 0.0001, or P < 0.01). But, DMF had no effect on activities of serum GPT and LDH (P > 0.05). CONCLUSION: DMF is a potent induction agent for QR and GSTs in the gland stomach, liver, lung and kidney of rats, which suggests it will hopefully be a protective and antidotic agent against human antioxidant injury.

Distinct temporal patterns of defensin mRNA regulation during drug-induced differentiation of human myeloid leukemia cells.

Defensins are microbicidal peptides and the principal constituents of neutrophil primary granules. They are presumed to play a prominent role in innate host defenses. We examined defensin mRNA levels during drug-induced differentiation of the promyelocytic leukemia cell line, HL-60. Transcription was restricted to promyelocyte, myelocyte, and very early metamyelocyte stages of the granulocytic pathway. Complete downregulation occurred during late granulocytic maturation or early during phorbol ester-promoted differentiation along the monocyte/macrophage lineage. Retinoic acid (RA) was the strongest inducer of defensin mRNA accumulation, even at doses too low to effect morphologic changes; the initial (first 48 hours), gradual increase resulted from transcriptional activation and was enhanced by granulocyte colony-stimulating factor. In contrast, addition of hybrid polar compounds led to a transient, drug-specific downregulation within the same time period, apparently by means of selectively induced, biphasic degradation of transcripts. Subsequent increase in transcript levels was faster and more pronounced with hexamethylene bisacetamide, relative to dimethyl sulfoxide (DMSO). DMSO-promoted effects were strikingly different in serum-free medium or in the presence of the tyrosine kinase inhibitor, genistein. Under these conditions, and although differentiation was unaffected, early defensin mRNA downregulation was final. The effect did not occur with RA and expression of other myeloid-specific genes was also unchanged. Addition of selected cytokines caused a similar "dip," only at earlier times and uncoupled from differentiation. Tumor necrosis factor-alpha markedly induced defensin levels after 2 days in previously untreated HL-60 cells, but inhibited expression in RA-differentiated cells. These results begin to detail a complex regulation of defensin mRNA synthesis with both spatial and temporal control elements, and a unique modulation by chemical agents, cytokines, and serum-factors.

Characterization of the inhibitory effects of transforming growth factor-beta on a human colon carcinoma cell line.

The effects of transforming growth factor-beta (TGF beta) on a human colon carcinoma cell line (MOSER) were investigated. TGF beta, at low concentrations (between 0.1 and 1.0 ng/ml), inhibited the proliferation of MOSER cells both in monolayer culture and soft agarose, in a dose-dependent manner. MOSER cells adapted to growth in chemically defined serum-free medium were more sensitive to the inhibitory effects of TGF beta than cells maintained in serum-supplemented medium. Morphological changes in MOSER cells, observed with TGF beta, were similar to those seen with the chemical differentiation agent N,N-dimethylformamide. Also in similarity to the effects of N,N-dimethylformamide, TGF beta induced a time- and concentration-dependent increase in soluble extracellular fibronectin. Binding studies with [125I]TGF beta revealed a relatively low number of binding sites on MOSER cells (13%) compared with mouse embryo fibroblastic (AKR-2B) cells. Thus far, other colon carcinoma cell lines, some displaying TGF beta receptors, have been reported to be unresponsive to TGF beta. This study is therefore the first to demonstrate a TGF beta-responsive colon carcinoma cell line.

N,N-dimethylformamide-induced synthesis of an anti-fibronectin reactive protein in cultured human colon carcinoma cells.

The cell surfaces of human colon cancer cells before and after exposure to N,N-dimethylformamide (DMF) were probed using radioiodination and immunofluorescent labeling techniques. Growth of the human colon carcinoma cell line HCT MOSER in DMF-supplemented culture medium resulted in monolayer culture growth and marked cell morphology alterations consisting of cellular flattening and elongation. Accompanying the morphology alterations were distinct changes in the cell surface protein composition as determined by 125I labeling and electrophoresis. The cell surface changes associated with growth of HCT MOSER cells in the presence of DMF were dependent upon time of exposure to DMF and DMF concentration. Furthermore, removal of DMF-treated HCT MOSER cells from DMF-containing growth medium caused reversion of both cell morphology and cell surface composition to a state comparable to that of cells not exposed to DMF. The HCT MOSER cell surface alterations produced by DMF included a reduction of radioiodinated surface proteins with molecular weights of 87,000, 120,000, and 180,000 and an increase of a 125I-labeled surface protein with a molecular weight of 200,000-250,000. Appearance of a surface protein of approximately 200,000 molecular weight and assumption of a fibroblast-like morphology by DMF-treated HCT MOSER cells suggested that this approximately 200,000 molecular weight material might be fibronectin. Immunofluorescent labeling with anti-human fibronectin showed that HCT MOSER cells grown in DMF did manifest an anti-fibronectin immunoreactive material that was only transiently associated with the cell surface before being released. DMF-treated HCT MOSER cultures continued to express surface carcinoembryonic antigen, indicating that the presence of material immunoreactive with anti-human fibronectin was not secondary to proliferation of a contaminating fibroblast population. The response of HCT MOSER cells to DMF paralleled in many ways that previously reported for methylcholanthrene-transformed AKR-2B (AKR-MCA) fibroblasts. However, unlike AKR-MCA cells, HCT MOSER cells did not exhibit an increase in 125I incorporation per microgram DNA as a function of time of exposure to DMF, which suggests that the surface protein with a molecular weight of approximately 200,000 induced by DMF was not retained on the cell surface.