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The high lysability by LAK cells of colon-carcinoma cells resistant to doxorubicin is associated with a high expression of ICAM-1, LFA-3, NCA and a less-differentiated phenotype.

A human colon-carcinoma cell subline resistant to doxorubicin (LoVo/Dx), previously shown to be more lysed than the chemosensitive subline LoVo/H by different immune effectors, is reported here to be similarly susceptible to direct, anti-proliferative effect of soluble cytokines (TNF-alpha and/or IFN-gamma). More adhesion molecules ICAM-1, LFA-3 and NCA were expressed on LoVo/Dx than on LoVo/H, while no significant amounts of CEA were detectable on the cell surface or in culture supernatant of either tumor subline. Anti-ICAM-1, anti-LFA-3 and anti-NCA monoclonal antibodies (MAbs) caused a marked reduction of lysis by interleukin-2 (IL-2) activated lymphocytes (LAK) of LoVo/Dx, whereas a lower effect was evident on LoVo/H. A pool of these antibodies was able to further increase the inhibition of the LAK lysis of both sublines. LoVo/Dx displayed a less differentiated phenotype as assessed by morphology, in vitro growth and altered or increased expression of markers such as desmoplakin and vimentin respectively, and disappearance of mucin. Treatment of LoVo sublines with differentiating agents (dimethylformamide and retinoic acid) led to a decreased expression of all adhesion molecules studied, accompanied by increased resistance to LAK-mediated lysis. These data indicate that sensitivity of chemoresistant tumor cells to cytotoxic effectors depends on the level of expression of adhesion molecules, including NCA, and is related to differentiation stage.

Antigens, CD↗

Surface modification of metallic cardiovascular stents by strongly adhering aliphatic polyester coatings.

This article reports on a novel two-step strategy for the coating of cardiovascular stents by strongly adhering biocompatible and biodegradable aliphatic polyesters. First, a precoating of poly(ethylacrylate) (PEA) was electrografted onto the metallic substrate by cathodic reduction of the parent monomer in dimethylformamide (DMF). The electrodeposition of PEA, in a good solvent of it, was confirmed by both Infra-red and Raman spectroscopies. The pendant ester groups of PEA were then chemically reduced into aluminum alkoxides, able to initiate the ring-opening polymerization (ROP) of either D,L-lactide (LA) or epsilon-caprolactone (CL). Growth of biodegradable PLA or PCL coatings from the adhering precoating was confirmed by both Infra-red and Raman spectroscopies, and directly observed by scanning electron microscopy (SEM). This type of coating can act as an anchoring layer for the subsequent casting of drug-loaded polyester films allowing the controlled release of antiproliferative agents for the treatment of in-stent restenosis.

Biocompatible Materials↗

Biodegradable porous polyurethane scaffolds for tissue repair and regeneration.

Critical-size bone defects usually require the insertion of autogenous bone graft to heal. Harvesting of bone is traumatic and results in high morbidity at the donor site. A potential alternative to bone graft may be a bone substitute with adequate biocompatibility and biological properties produced from ceramics or bioresorbable/biodegradable polymers. In the present study, new elastomeric biodegradable polyurethanes with an enhanced affinity toward cells and tissues were synthesized using aliphatic diisocyanate, poly(epsilon-caprolactone) diol, and biologically active 1,4:3,6-dianhydro-D-sorbitol (isosorbide diol) as chain extender. The polymers were processed into 3D porous scaffolds by applying a combined salt leaching-phase inverse process. The critical parameters controlling pore size and geometry were the solvents and nonsolvents used for scaffold preparation and the sizes of the solid porogen crystals. Scaffolds prepared from the polymer solution in solvents such as dimethylsulfoxide or methyl-2-pyrrolidone did not have a homogenous pore structure. Many pores were interconnected, but numerous pores were closed. Irrespective of the high pore-to-volume ratio (75%), the scaffolds showed poor water permeability. The best solvent for the preparation of scaffolds from the polyurethane used in the study was dimethylformamide (DMF). The type of nonsolvent admixed to the polymer solution in DMF strongly affected the scaffolds' pore structure. The elastomeric polyurethane scaffold prepared from the optimal solvent-nonsolvent mixture had regular interconnected pores, high water permeability, and a pore-to-volume ratio of 90%. The osteoconductive properties of the 3D porous polyurethane scaffolds can be additionally promoted by loading them with calcium phosphate salts such as hydroxyapatite or tricalcium phosphate, thus making them promising candidates for bone graft substitutes.

Absorbable Implants↗

Preparation and magnetic resonance imaging effect of polyvinylpyrrolidone-coated iron oxide nanoparticles.

Polyvinylpyrrolidone (PVP)-coated iron oxide nanoparticles were prepared by the thermal decomposition of Fe(CO)(5) (iron pentacarbonyl) in one step. X-ray diffraction (XRD), transmission electron microscopy (TEM), electrophoretic light scattering (ELS), infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) together with the variation of the molar ratio of PVP/Fe(CO)(5), solvent, and molecular weight of PVP, were used to characterize the PVP-coated iron oxide nanoparticles. Fifty to hundred nanometer-sized iron oxide nanoclusters with a spherical shape were formed in dimethylformamide (DMF), used as a solvent, and exhibited an enhanced stability in the aqueous media. Their magnetic properties were investigated by superconducting quantum interface device (SQUID). The in vitro cytotoxicity test revealed that the PVP-coated iron oxide nanoparticles exhibited excellent biocompatibility by MTT assay. Magnetic resonance imaging (MRI) effect was observed with the administration of PVP-coated iron oxide nanoparticles through the marginal vein of rabbit, resulting in improved detection of the liver lesions.

Coated Materials, Biocompatible↗

An AM1 semiempirical study of host-guest complexation in hemicarcerand complexes.

Semiempirical AM1 calculations have been carried out on host-guest complexes of model hemicarcerands 1a and 2a. The justification for the choice of the AM1 Hamiltonian was based on a comparison between reported X-ray data for the smaller tetrabromocavitand 4a and computational results obtained using several different Hamiltonians. The complexation behavior of hemicarcerands 1a and 2a have been compared with experimental results reported by Cram et al. for the related hemicarcerands 1b and 2b. Based on this comparison, a criterion for predicting guest encapsulation was developed, E(complexation), which relies on the calculation of AM1 heats of formation for host, guest, and hemicarceplex. If E(complexation) is lower than 10 kcal/mol, then a guest will be encapsulated, while if it is greater than 30 kcal/mol, a guest will not be encapsulated. The use of constrained-path AM1 optimizations to determine the energy barriers to guest entry and exit from the host was found to be a useful tool for examining suitable host-guest combinations when the E(complexation) criteria does not hold. We have computed the barriers to exit of N, N-dimethylformamide (dmf) and furan from the hemicarcerand 1a, the former has been compared with the experiment and shows excellent agreement. Based on the success of the above computational methods in predicting which host-guest combinations will form stable hemicarceplexes we have synthesized a new target hemicarceplex 1b.furan.

Journal Article↗

Procaine inhibits the erythroid differentiation of MEL cells by blocking commitment: possible involvement of calcium metabolism.

The action of procaine on the terminal erythroid differentiation of murine erythroleukemia (MEL) cells has been investigated at the level of individual cells. At concentrations (7 X 10-4 M) which had no inhibitory effect on cell growth, pretreatment of these cells with procaine for 12-24 hr caused a pronounced inhibition ( greater than 90%) of commitment of terminal erythroid differentiation of dimethyl sulfoxide (DMSO)-treated cells. Simultaneous treatment of MEL cells with DMSO and procaine, however, resulted to only slight inhibition (less than 20%) of commitment. Blockade of commitment by procaine pretreatment appears to be general since it was observed in cells treated with other inducers (6-thioguanine, dimethylformamide). Procaine pretreatment did not abolish the ability of MEL cells to complete the "latent period" and commit upon the removal of the block. Reversal of procaine inhibition of commitment was obtained by the addition of either CaCl2 (1.0 mM), calcium ionophore A23817 (1 microgram/ml), but not of MgCl2 (1.0 mM). From these data we conclude that procaine inhibits the terminal erythroid differentiation of MEL cells by blocking an event or process required for commitment which occurs prior to commitment itself. Our results suggest that this process involves calcium metabolism.

Animals↗

Cyclic AMP-dependent and -independent protein kinases and protein phosphorylation in human promyelocytic leukemia (HL60) cells induced to differentiate by retinoic acid.

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8-bromo-cyclic adenosine 3:5 monophosphate (8-Br-cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate-dependent protein kinase (cyclic AMP-dPK), we investigated the role of cyclic AMP-dPK and adenosine 3:5 cyclic monophosphate-independent protein kinase (cyclic AMP-iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP-dPK and cyclic AMP-iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP-dPK and cyclic AMP-iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type l cytosolic cyclic AMP-dPK and cyclic AMP-iPK (protamine kinase) while no such increases are noted in cells treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8-azido-cyclic AMP demonstrate marked increases in type l cyclic AMP-dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA-differentiated cells. (4) The appearance and disappearance of specific cyclic AMP-dependent and -independent protein phosphorylations are associated with the induced myeloid differentiated state.

Carrier Proteins↗

Modulation of fibronectin, laminin, and cellular adhesion in the transformation and differentiation of murine AKR fibroblasts.

The functional relationship between membrane/cell surface expression of fibronectin and laminin and transformation/differentiation was examined in an AKR mouse fibroblastic cell model. This model consisted of the untransformed AKR-2B cells, their chemically transformed counterpart (AKR-MCA cells) and the chemically differentiated form of the AKR-MCA cells. The transformed AKR-MCA cells were found to express more surface laminin and less fibronectin than the untransformed AKR-2B cells. The transformed AKR-MCA cells were slower to attach and spread on both plastic and type IV collagen-coated dishes in comparison to the AKR-2B cells. However, a higher percentage of the AKR-MCA cells ultimately attached and spread on the type IV collagen-coated dishes. The induction of differentiation in the AKR-MCA cells by N,N-dimethylformamide (DMF) restored fibronectin to the surface of the AKR-MCA cells but reduced laminin expression only slightly. The DMF-treated AKR-MCA cells resembled the AKR-2B cells in that they rapidly attached and spread on plastic dishes and dishes coated with type IV collagen. They also resembled the AKR-MCA cells in that a high proportion ultimately attached and spread on the collagen-coated dishes.

Animals↗

Kidney-specific enzyme expression by human kidney cell lines generated through oncogene transfection.

The human kidney cell line 293 was generated by transfection of adenovirus DNA into normal human embryonic kidney (HEK) cells (Graham et al., 1977), whereas the human kidney cell lines ST-1i and STt-4i were generated by transfection of HEK cells with plasmids encoding SV40 viral oncogenes (Abcouwer et al., 1989). In this study, we examined kidney-specific enzyme activity levels in 293, ST-1i, and STt-4i cells to determine their ability to exhibit kidney-specific gene expression. Enzymes examined were leucine aminopeptidase (LAP), gamma-glutamyl transpeptidase (gamma-GTP), and the disaccharidases trehalase and maltase. Enzymatic activity levels were compared to three other kidney cell lines (MDCK, OK, and LLC-PK1) as well as to normal human embryonic kidney (HEK) cells and the human hepatoma cell line, Hep G2. Modulation of kidney-specific enzyme activities was assessed in response to several differentiation-inducing agents (adenosine, n-butyric acid, hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), isobutyl methyl xanthine (IBMX), di butyryl cAMP, and retinoic acid). ST-1i and STt-4i exhibit elevated levels of LAP, gamma-GTP, trehalase, and maltase, consistent with their kidney cell origin, whereas 293 cells exhibit elevated levels of just gamma-GTP and maltase. Maltase and gamma-GTP enzyme activities in ST-1i and STt-4i cells were very responsive to the various inducing agents; 293 cells were less responsive at the inducer concentrations examined. None of the three human cell lines formed domes under any of the experimental conditions. In summary, ST-1i and STt-4i are comparable to normal HEK cells in expression of kidney-specific enzymes and in responsiveness to differentiation-inducing agents, in spite of continued expression of SV40 oncogenes.

Animals↗

Control of AKR fibroblast phenotype by fibronectin: regulation of cell-surface fibronectin binding receptor by fibronectin.

Results of previous studies show that the expression of fibronectin and its cell-surface fibronectin binding receptor is coregulated in 3-methylchloranthrene transformation of normal AKR-2B cells to form AKR-MCA cells and in N,N,-dimethylformamide (DMF) induction of differentiation of transformed AKR-MCA cells (1990, J. Cell. Physiol., 143:445). In this study, we tested the coregulation hypothesis by transfection experiments using an antisense fibronectin expression vector. We determined the effect of antisense fibronectin RNA expression on untransformed AKR-2B cells, and on the responses of transformed AKR-MCA cells to DMF treatment. Expression of antisense fibronectin RNA in AKR-2B cells down-modulated fibronectin production, reduced adhesion to extracellular fibronectin, and altered cellular morphology. Saturation binding and Scatchard analyses using radiolabelled fibronectin revealed a concurrent down-modulation of cell-surface fibronectin binding sites, but the binding affinity of the receptor for the ligand was not affected. Immunoblotting and immunostaining revealed down-modulation of the expression of alpha 5 beta 1 integrins. Expression of antisense fibronectin RNA in AKR-MCA cells down-modulated the ability of DMF to restore normal fibronectin production, cell-surface fibronectin binding receptor, adhesion to extracellular fibronectin, and cellular morphology. These studies show that both fibronectin and its cell-surface fibronectin binding receptor were tightly regulated during transformation and induction of differentiation in these cells, that the ligand and its cell-surface fibronectin binding receptor worked together to bring about phenotypic changes, and that fibronectin production regulated the expression of its cell-surface fibronectin binding receptor.

Animals↗

Improved cryoprotection and freeze-substitution of embryonic quail retina: a TEM study on ultrastructural preservation.

Conditions for cryofixation and freeze-substitution crucial to the ultrastructural preservation of embryonic quail retina were improved. As freeze-substitution makes gentle dehydration and chemical fixation of tissue possible, the suitability of different cryoprotectants were tested in the preceding cryofixation. Additionally, different conditions for chemical prefixation were studied. In cryofixation, all of the "classic" cryoprotectants caused more or less severe tissue destruction. Only dimethylformamide (DMF) and--with certain reservations--dimethylsulfoxide (DMSO) yielded improved structure preservation. Perfusion fixation with a mixture of formaldehyde/glutaraldehyde (FA/GA) was superior to GA alone. In comparison to conventional fixation and dehydration methods, freeze-substitution yielded better ultrastructural preservation of the embryos with fewer artifacts.

Animals↗

Mycobacterium sp., Rhodococcus erythropolis, and Pseudomonas putida behavior in the presence of organic solvents.

This work aimed at studying the behavior and tolerance of Mycobacterium sp. NRRL B-3805, Rhodococcus erythropolis DCL14 and Pseudomonas putida S12 cells in the presence of various concentrations of water miscible (ethanol, butanol, and dimethylformamide, up to 50% v/v) and water immiscible solvents (dodecane, bis(2-ethylhexyl) phthalate and toluene, up to 5% v/v). When incubated in the presence of these solvents, the cells were found to have lower tolerance to butanol and toluene than to the remaining solvents. Nevertheless, the concentrations of solvents endured by the tested strains show that they are quite solvent-tolerant, confirming their potential as biocatalysts in nonconventional systems. Microscopic observation of samples showed that the hydrophobic Mycobacterium sp. and R. erythropolis cells were able to aggregate to protect the population under stress conditions. Comparison of the results obtained at the single cell level by fluorescence microscopy and colony development on agar plates indicated that the primary effects of most solvents tested were on the cell membrane and replicating capability of the cells.

Cell Aggregation↗

Interaction of the peptide CF3-Leu-Ala-NH-C6H4-CF3 (TFLA) with porcine pancreatic elastase. X-ray studies at 1.8 A.

The peptide trifluoroacetyl-Leu-Ala-(p-trifluoromethylanilide), is a reversible inhibitor of pancreatic porcine elastase and is characterized by a Km of 2.5 x 10(-8) M. Co-crystals of the 1:1 complex were obtained in an acetate buffer + dimethylformamide solution at pH 5.7. Diffraction data were recorded on films at the LURE synchrotron facility. The inhibitor was localized on difference Fourier maps, and the refinement of the structure was performed by simulated annealing (XPLOR). The current agreement factor is R = 19% (for 13224 observed structure factors and 1.8 A effective resolution). The RMS deviations from ideality of bond distances and angles are 0.02 A and 2 degrees, respectively. The inhibitor molecule was found in the active site, bent around the side chain of Phe-215 in a geometry that resembles the previously reported structure of the CF3-Lys-Ala complex at 2.5 A, in a parallel beta-sheet association with the loop 214-216. The analysis of the close contacts (less than 3.5 A) indicates that the trifluoromethylamide bond interacts with the active site and not the Leu-Ala or Ala-anilide bonds. The two fluorinated groups of the inhibitor exhibit different specificities: the trifluoroacetyl group (N terminus) is tightly stacked between the two chain loops 191-195 and 213-215, while the trifluoromethylanilide (C terminus) shows less specificity and only a single contact.

Animals↗

An automated parallel crystallisation search for predicted crystal structures and packing motifs of carbamazepine.

An automated parallel crystallisation search for physical forms of carbamazepine, covering 66 solvents and five crystallisation protocols, identified three anhydrous polymorphs (forms I-III), one hydrate and eight organic solvates, including the single-crystal structures of three previously unreported solvates (N,N-dimethylformamide (1:1); hemi-furfural; hemi-1,4-dioxane). Correlation of physical form outcome with the crystallisation conditions demonstrated that the solvent adopts a relatively nonspecific role in determining which polymorph is obtained, and that the previously reported effect of a polymer template facilitating the formation of form IV could not be reproduced by solvent crystallisation alone. In the accompanying computational search, approximately half of the energetically feasible predicted crystal structures exhibit the C=O...H--N R2(2)(8)dimer motif that is observed in the known polymorphs, with the most stable correctly corresponding to form III. Most of the other energetically feasible structures, including the global minimum, have a C=O...H--N C(4) chain hydrogen bond motif. No such chain structures were observed in this or any other previously published work, suggesting that kinetic, rather than thermodynamic, factors determine which of the energetically feasible crystal structures are observed experimentally, with the kinetics apparently favouring nucleation of crystal structures based on the CBZ-CBZ R2(2)(8) motif.

Algorithms↗

Differentiating nonaqueous titration of aspirin, acetaminophen, and salicylamide mixtures.

Mixtures containing aspirin, acetaminophen, and salicylamide were assayed potentiometrically by nonaqueous titration. The difference in pKa values for these weak acids was sufficient to permit successful differentiation. The titrant was tetrabutylammonium hydroxide, and the titration solvent was dimethylformamide. The procedure was applied to commercial dosage forms.

Acetaminophen↗

High-speed liquid chromatographic analysis of sulfasalazine (salicylazosulfapyridine).

A high-speed liquid chromatographic method for analysis of sulfasalazine (salicylazosulfapyridine) in bulk powder and tablet dosage form is presented. Analysis is accomplished with a reverse-phase partition column and 10% 2-propanol in pH 7.7 phosphate buffer as the mobile phase. The method of analysis utilizes a simple, one-step, solubilization procedure with dimethylformamide, addition of an internal standard, and chromatography. The method is specific for sulfasalazine in the presence of starting materials, degradation products, or by-products from its manufacture.

Chromatography, High Pressure Liquid↗

Effect of solvent on tetrazolium reaction.

The rate of color development of a tetrazolium formazan is shown to be inversely proportional to the dielectric constant of the solvent medium and directly proportional to the hydrogen-bonding capability of solvent mixtures having the same dielectric constant. The geometric isomers of the formazans have different absorbance maxima, and the wavelength of maximum absorbance of a mixture of formazans in different solvents depends upon which isomer predominates in that solvent. The trans-syn-isomer (blue form) of blue tetrazolium has a maximum absorbance at 625 nm in dimethylformamide while the trans-anti-isomer (red form) absorbs at 517 nm in methanol. The absorbance maxima of the corresponding isomers of the formazans of triphenyltetrazolium occur at 535 and 485 nm, respectively. Water and/or methanol (to a lesser extent) are important in the stabilization of the trans-anti-isomer, since the small size of these two substances allows them to form strong intermolecular hydrogen bonds with one or both nitrogen atoms of the azo linkage, thereby preventing the formation of the intramolecular hydrogen bonding exhibited by the trans-syn-isomer. The formazan produced by the reaction of corticosteroids with tetrazolium in strongly basic media can lose a reduction unit and be reoxidized to the tetrazolium. This reaction is solvent dependent and occurs at a much faster rate in chloroform than in alcohol USP.

Adrenal Cortex Hormones↗

Liquid chromatography in pharmaceutical analysis VI: determination of dantrolene sodium in a dosage form.

Operating conditions are described for the qualitative and quantitative determination of dantrolene sodium by high-pressure liquid chromatography. A 10-mum porous silica column was employed, using carbon tetrachloride-dimethylformamide (90:10) as the mobile phase. The flow rate was 2.0 ml/min (1800 psig), and the peaks were detected at 375 nm. The analysis of a dosage form can be carried out within 30 min with an accuracy of 3.1%. The results agree favorably with those obtained with a modified spectrophotofluorometric method.

Capsules↗