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PubMed1995

Physicochemical characterization of a new respirable form of nedocromil.

Elongated, respirable particles of nedocromil were prepared by its crystallization from N-methyl-2-pyrrolidone (NMP) obtained from an aqueous solution of its sodium salt by precipitation with hydrochloric acid. Aerosols of these particles were recently generated and characterized for their aerodynamic properties (Chan, H.-K.; Gonda, I. J. Aerosol Med. 1993, 6, 241-249). This paper reports the physiochemical properties of the solid form of nedocromil as characterized by polarizing optical and scanning electron microscopies, thermal analysis (DSC, TGA), X-ray powder diffraction, water vapor adsorption isotherms, mass spectrometry, solid state Fourier transform IR and solution 1H-NMR spectroscopies.

Administration, Inhalation↗
PubMed2004

Design of temperature sensitive imprinted polymer hydrogels based on multiple-point hydrogen bonding.

Weakly cross-linked temperature sensitive imprinted polymer hydrogels that recognize L-pyroglutamic acid (Pga) molecules via multiple-point hydrogen bonding were designed and synthesized. The amount of adsorption for Pga in imprinted hydrogels is 3-4 times higher than that in non-imprinted hydrogels. The selectivity test of imprinted polymer gels was carried out by using a series of structurally related compounds Pga, pyrrolidine, 2-pyrrolidone, L-proline as substrates. The results show that imprinted polymer gels exhibit high selectivity for Pga as compared to all the other tested substrates. The imprinted polymer hydrogels show good temperature sensitivity, special selectivity and reusability, suggesting that the polymer hydrogels would have an enormous potential for application in controlled drug release and separation field.

Adsorption↗
PubMed2006

Fibrous membranes electrospinning from acrylonitrile-based polymers: specific absorption behaviors and states of water.

Fibrous membranes with a fiber diameter ranging from 80 to 800 nm are prepared from polyacrylonitrile and poly[acrylonitrile-co-(N-vinyl-2-pyrrolidone)] by the electrospinning process. The parameters can be controlled to fabricate fibrous membranes with similar fiber diameters (between 600 and 800 nm) for further studies on the swelling behaviors and water states. Water swelling experiments indicate that the fibrous membrane has a great capacity for water sorption, which reaches a maximum in a few minutes because of its extremely high porosity. Furthermore, a remarkable overshoot occurs as a result of polymer chain relaxation and the non-compact structure of the fibrous membranes. Contrary to the dense membrane, the equilibrium water content in the fibrous membrane decreases with the content of hydrophilic NVP though the maximum is almost the same. Results from DSC experiments demonstrate that only non-freezable bound water and free water can be distinguished in the fibrous membrane. On the basis of the results of water swelling and DSC experiments, it is concluded that the specific behaviors of the fibrous membranes are induced by the non-compact and pore-fiber discontinuous structure, which is different from either dense membranes or hydrogels. [GRAPHS: SEE TEXT] DSC curves of fully swollen electrospun fibrous membranes and of fully swollen dense membranes with different NVP contents.

Absorption↗
PubMed2001

Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has been applied successfully in the treatment of male infertility in humans and in fertilization research in mice. However, the technique has had limited success in producing offspring in other species including the rabbit. The aim of this research was to test the in vitro and in vivo developmental of rabbit oocytes after ICSI. Sperm used for ICSI were collected from mature Dutch Belted buck and washed 2-3 times with PBS +0.1% polyvinyl alcohol (PVA) and then mixed with 10% polyvinyl pyrrolidone (PVP) prior to microinjection. Oocytes were collected from superovulated does 14-15 hr after hCG injection and were fertilized by microinjection of a single sperm into the ooplasm of each oocyte without additional activation treatment. After ICSI, the presumed zygotes were either cultured in KSOM +0.3% BSA for 4 days or transferred into oviducts of recipient does at the pronuclear or 2-cell stage. A high percentage of fertilization (78%, n = 114) and blastocyst development (39%) was obtained after ICSI. Control oocytes, receiving a sham injection, exhibited a lower activation rate (31%, n = 51) and were unable to develop to the blastocyst stage, suggesting that the blastocysts developed following ICSI were derived from successful fertilization rather than parthenogenetic development. A total of 113 embryos were transferred to six recipient does. Two recipients became pregnant and delivered seven live young. Our results demonstrated that rabbit oocytes can be successfully fertilized and activated by ICSI and can result in the birth of live offspring.

Animals↗
PubMed2002

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.

Animals↗
PubMed2004

Development and evaluation of biocompatible inks for the local measurement of oxygen using in vivo EPR.

In vivo EPR oximetry is a powerful minimally invasive method that allows the measurement of oxygen in tissues through the use of a paramagnetic probe. In the present study, we investigated new strategies for preparing biocompatible inks containing carbon black particles (Printex U), which could be used as oxygen sensors. The carbon black particles were dispersed in solutions of biocompatible polymers of carboxy methyl cellulose (CMC), hydroxypropyl methyl cellulose (HPMC) or polyvinyl pyrrolidone (PVP). A total of 12 polymers with different molecular weights were tested. A physico-chemical characterization of the inks was carried out to assess the sedimentation of the particles, the rheological behavior of these inks, and the relative diffusion of the inks. The preparations with CMC and PVP had the highest viscosity and stability. The presence of the polymers did not modify the calibration curves (EPR linewidth as a function of the pO2) of the carbon black. In vivo, the oxygen sensors were stable for at least one month in muscles as the EPR linewidth remained fully sensitive to induced ischemia or carbogen challenge. The calibration curve was not modified after this period of implantation. A first study of biocompatibility was carried out in vitro (hemolysis and cytotoxicity assay) and in vivo (histological examination). No sign of toxicity was observed using these inks. These preparations are good candidates for future in vivo studies including clinical trials.

Animals↗
PubMed1995

The structure of human pancreatic alpha-amylase at 1.8 A resolution and comparisons with related enzymes.

The structure of human pancreatic alpha-amylase has been determined to 1.8 A resolution using X-ray diffraction techniques. This enzyme is found to be composed of three structural domains. The largest is Domain A (residues 1-99, 169-404), which forms a central eight-stranded parallel beta-barrel, to one end of which are located the active site residues Asp 197, Glu 233, and Asp 300. Also found in this vicinity is a bound chloride ion that forms ligand interactions to Arg 195, Asn 298, and Arg 337. Domain B is the smallest (residues 100-168) and serves to form a calcium binding site against the wall of the beta-barrel of Domain A. Protein groups making ligand interactions to this calcium include Asn 100, Arg 158, Asp 167, and His 201. Domain C (residues 405-496) is made up of anti-parallel beta-structure and is only loosely associated with Domains A and B. It is notable that the N-terminal glutamine residue of human pancreatic alpha-amylase undergoes a posttranslational modification to form a stable pyrrolidone derivative that may provide protection against other digestive enzymes. Structure-based comparisons of human pancreatic alpha-amylase with functionally related enzymes serve to emphasize three points. Firstly, despite this approach facilitating primary sequence alignments with respect to the numerous insertions and deletions present, overall there is only approximately 15% sequence homology between the mammalian and fungal alpha-amylases. Secondly, in contrast, these same studies indicate that significant structural homology is present and of the order of approximately 70%. Thirdly, the positioning of Domain C can vary considerably between alpha-amylases. In terms of the more closely related porcine enzyme, there are four regions of polypeptide chain (residues 237-250, 304-310, 346-354, and 458-461) with significantly different conformations from those in human pancreatic alpha-amylase. At least two of these could play a role in observed differential substrate and cleavage pattern specificities between these enzymes. Similarly, amino acid differences between human pancreatic and salivary alpha-amylases have been localized and a number of these occur in the vicinity of the active site.

Amino Acid Sequence↗
PubMed2005

Alpha- and beta- aspartyl peptide ester formation via aspartimide ring opening.

The undesirable reaction of aspartimide formation has been proved to occur under both acid and base conditions in solid-phase peptide synthesis and is dependent on the beta-carboxyl protecting group, the acid or base used during the synthesis, as well as the peptide sequence. The hydrolysis of aspartimide-containing peptides, especially during HPLC purification, yields a mixture of alpha- and beta-aspartyl peptides that can not be purified easily. A previous study demonstrated that treatment of aspartimide-containing peptides with methanol in the presence of 2% diisopropylethylamine in solution leads to alpha- and beta-aspartyl peptide methyl esters. Taking advantage of these results and aiming at elucidating the optimal conditions for aspartimide ring opening, the effect of different types and concentrations of alcohols (primary and secondary) and bases (diisopropylethylamine, collidine, 4-pyrrolidinopyridine, 1-methyl-2-pyrrolidone, piperidine and KCN) was tested at various temperatures and reaction times. The best results were obtained with a combination of a primary alcohol and diisopropylethylamine, while aspartimide ring opening by secondary alcohols occurred only at high temperatures. The optimal conditions were also applied to solid-phase peptide synthesis.

Amino Acids↗
PubMed2007

Development of a liquid chromatography/electrospray ionization tandem mass spectrometric method for the determination of hydroxyl radical.

A rapid method combining liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was developed for the determination of the hydroxyl radical (.OH). .OH generated via Fenton reaction was spin-trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and then analyzed by LC/ESI-MS/MS in multiple reaction monitoring (MRM) mode, using N-methyl-2-pyrrolidone (NMP) as the internal standard. The peak area ratio of DMPO-OH to NMP positively correlated with the concentration of .OH. The relative standard deviation (RSD) of the method was 1.13% (n = 8). The present method was successfully applied to evaluate the .OH scavenging capacity of several phenolic acids.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Investigation of human hair fibers using lateral force microscopy.

Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used to investigate the morphologic and surface changes associated with various surface modifications to human hair. These included extraction with a series of solvents, bleaching, and treatment with a cationic copolymer. The study assessed the ability of these techniques to distinguish the changes in surface properties, including morphology and friction coefficient, as manifested in changes brought about by the indicated surface modifications. While topographic morphology can easily be investigated with contact AFM. LFM offers an additional tool for probing the surface distribution of oils and waxes. The removal of surface lipids from the fiber surface was accomplished using soxhlet extraction with t-butanol and n-hexane, while the free internal lipids (within the fiber structure) were removed by extraction with a mixture of chloroform and methanol (70:30, v/v). In addition, the surface of hair was modified with the cationic polymer, co(vinyl pyrrolidone-methacrylamidopropyl trimethylammonium chloride [PVP/MAPTAC]), and its distribution on the surface was monitored. Ambient AFM and LFM studies of surface modified and native fibers clearly indicate that when investigated as a function of tip loading force, the different modifications result in changes of the friction coefficient, which increase in this order: native, bleached, solvent extracted, and polymer-treated hair. Friction images show surface variations that are interpreted as areas of varying lipid film coverage. In addition, topographic images of the fibers show the presence of small pores, which become increasingly prevalent upon solvent extraction.

Chloroform↗
PubMed1994

Application of polymer-protected ultrafine platinum particles to the immunological detection of human serum albumin.

The immunological properties of ultrafine platinum particles protected with poly(methyl acrylate)-co-N-vinyl-2-pyrrolidone, having a group chemically reactive with protein, were examined. Polymer-protected ultrafine particles were able to chemically bond multiple human serum albumin (HSA) molecules on their surface. The particles behaved as a multi-epitope-type antigen for the immunoreaction. In an ELISA, the HSA-bound particles and HSA molecules competitively reacted against the anti-HSA monoclonal antibodies. The limit of detection of HSA was ca. 10 ng/ml in the assay. The particles were used in an immunodot analysis of anti-HSA polyclonal antibody. Within 30 min of the reaction, 1-10 micrograms/ml of HSA was detected. The protein fixed polymer-protected ultrafine particles are a useful new material in immunochemistry.

Animals↗
PubMed1995

A simple serum-free freezing medium for serum-free cultured cells.

Because the presence of serum in cell culture raises safety problems for the production of biologicals, we have developed a serum-free medium (SFM) for the cryopreservation of animal cells. This medium is based of the SFM MDSS2, to which 10% dimethylsulfoxide (DMSO) and 0.1% methylcellulose or 3% polyvinyl pyrrolidone or no other additive than DMSO were added. Both, Vero and BHK-21 cells regularly cultivated in MDSS2, could be cryopreserved in the three serum-free freezing media and could be thawed without any cell loss. No differences could be found between the cells in the standard freezing medium (DMEM containing 10% fetal calf serum and 10% DMSO) and those frozen in SFM, with respect to cell growth and viability. In the case of the Vero cells no differences were observed with respect to their attachment. This medium represents the last step in fulfilling a complete serum-free animal cell based bioprocess.

Animals↗
PubMed1994

Cryoprotection of yeast by alcohols during rapid freezing.

We have investigated the cryoprotective effect of alcohols on Saccharomyces cerevisiae and other yeast under various freezing conditions. For S. cerevisiae, at a cooling rate of 3 degrees C min-1 methanol and ethanol acted as cryosensitizers. However, at a cooling rate of 200 degrees C min-1, both methanol and ethanol proved superior to all other cryoprotectants tested, including glycerol, dimethyl sulfoxide, lactose, trehalose, polyethylene glycol, and polyvinyl pyrrolidone. Propan-2-ol also demonstrated a small but measurable effect although propan-1-ol and butan-1-ol demonstrated no cryoprotective effect. A minimum cooling rate of 25 degrees C min-1 to elicit the cryoprotective effect of ethanol was necessary; below this rate it acted as a cryosensitizer. At cooling rates up to 650 degrees C min-1 substantial cryoprotective effect was still evident. Although the effect of ethanol was variable for other yeast genera tested, ethanol acted positively for all strains of S. cerevisiae. We hypothesize that the cryoprotective effect of alcohols during rapid cooling is a result of their ability to induce increased membrane permeability, allowing rapid water equilibration during extracellular freezing and avoidance of intracellular ice crystal formation.

Alcohols↗
PubMed2000

Effects of high-molecular-weight cryoprotectants on platelets and the coagulation system.

The objective of this study is to examine the effects of the most widely used high-molecular-weight cryoprotectants on the coagulation system. Dextran, hydryoxyethyl starch (HES), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), and albumin were added at different concentrations in the range between 0.01-1% (w/v) to solvent/detergent-treated plasma. Using a STA/STA Compact coagulation analyzer the following clotting tests were performed: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Factor V, and Factor VIII percentage of activity. PVP and PEG caused a significant increase in APTT, a decrease in Factor VIII percentage of activity, and a slight decrease in TT, while PT and Factor V percentage of activity remained unchanged. Dextran, HES, and albumin did not effect the clotting tests. The effect of high-molecular-weight cryoprotectants on platelets was assessed by platelet-induced clot retraction (PICR) and aggregation with thrombin and agglutination with ristocetin. Platelet aggregation and agglutination were unaffected by all cryoprotectants tested; however, PICR was significantly reduced in the presence of PVP or PEG. Possible mechanisms by which PVP and PEG interfere with the coagulation system are discussed. We also raise issues concerning the development of one-step blood cryopreservation techniques which do not require cryoprotectant removal prior to transfusion.

Albumins↗
PubMed1997

Generation of Light-Induced Electrical Potential from Ion Exchange Membranes Containing 4,4'-Bipyridine Moiety

The effect of various swelling solvents for anion exchange membranes having a viologen moiety as the anion exchange groups on photovoltage was examined to obtain a high photovoltage and to clarify the mechanism of charge transfer. Anion exchange membranes having a viologen moiety, based on a copolymer membrane of chloromethylstyrene and divinylbenzene and a film of chloromethylated polysulfone, were swelled with various solvents, and clamped between two ITO electrodes (indium-tin oxide electrodes), and a xenon lamp irradiated the membrane from one side. In general, the solvents that swelled the membranes well and provided a high photovoltage included ethylene glycols, 1,3-dioxolane, 1,3-dioxane, and 1-methyl-2-pyrrolidone. Especially, the membrane swelled with 1,3-dioxolane generated a high photovoltage. It is thought from these results that methylene groups neighboring oxygen and between two oxygens, which is an electron acceptor, are related to the charge transfer in the system. When the cell was assembled by sandwiching a cation exchange membrane (as a separator) between two anion exchange membranes with a viologen moiety, the photovoltage significantly decreased with an increase in repeat time of the irradiation and interruption. On the other hand, the voltage of the cell having of an anion exchange membrane separator did not significantly decrease with the same procedure. When non-ion-exchanged chloride ions were added to the cell, the generation of the photovoltage became high compared to the cell without the ions. It was concluded from these results that chloride ions also play an important role in the charge transfer in the cell.

Journal Article↗
PubMed1998

Encapsulation of Inorganic Particles by Dispersion Polymerization in Polar Media

Polymer encapsulation of small silica particles, using dispersion polymerization of styrene in aqueous ethanol medium with poly(N-vinyl pyrrolidone) (PVP) as stabilizer, is described. Silica particles, directly synthesized by the Stober process in an aqueous ethanol medium, are either unreacted (hydrophilic character) or coated with 3-(trimethoxysilyl)propyl methacrylate (MPS) (hydrophobic character), which is grafted at the silica particle surface. When the bare silica particles are used as the seed, there is a strong tendency of the silica beads to cover the surface of the polystyrene particles and obviously encapsulation does not occur. On the contrary, when the silica surface is made hydrophobic by coating, the inorganic particles are entirely contained in the polystyrene particles as evidenced by microscopy techniques (TEM, SEM, AFM). It is shown that some polystyrene chains are then chemically bonded to the silica particles, through the coupling agent MPS, and that only a small amount of bonded polystyrene, compared to the total polystyrene synthesized, is sufficient to obtain encapsulation of the silica particles with the entire amount of polystyrene synthesized during the polymerization. Under our experimental conditions, each polystyrene latex particle contains, on average, 4 to 23 silica beads depending, in particular, on the size of the silica. We believe that it is possible to control the composite particle size and morphology by a convenient choice of the composition of the system. Moreover, this new polymer-encapsulation process could be used to synthesize other organic-inorganic composite particles, using, for example, other monomers or minerals. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗
PubMed1998

Aggregation Behavior of Sugar Surfactants in Aqueous Solutions: Effects of Temperature and the Addition of Nonionic Polymers.

The aggregation behavior, critical micelle concentration (cmc) and micelle aggregation number (N), of dodecyl maltoside (DM), octyl glucoside (OG), and Hecameg has been investigated in water and in water plus one of the three water-soluble polymers, polyoxyethylene (POE), polyoxypropylene (POP), and polyvinyl pyrrolidone (PVP), by means of florescence probing and time-resolved fluorescence quenching. The cmc of DM in water increased with temperature and showed a slight increase in the presence of POE. The aggregation number N of DM micelles was nearly independent of concentration (0.25-1 wt %) and temperature (16-60 degreesC). It remained invariant upon addition of 2 wt % POE or PVP but decreased slightly upon addition of the more hydrophobic POP. With increasing temperature, the cmc of OG decreased, went through a shallow minimum at around 35 degreesC, and increased. Addition of POE slightly increased the cmc in the whole temperature range. The aggregation number of OG micelles showed a fairly flat maximum at around 30 degreesC, and was unaffected by the presence of 2 wt % POE or PVP. However, N showed a complex dependence on temperature in the presence of POP, with lower values than in pure water below 15 degreesC, and rapidly increasing quencher-dependent values above this temperature. Hecameg was characterized by N-values nearly independent of temperature and concentration. Intermicellar exchanges of probe and/or quencher were observed with OG and Hecameg, but not with DM. The above results are compared to those for the nonionic ethoxylated surfactants. The effect of various parameters on the micelle aggregation number, the micelle polydispersity, the occurrence of sugar surfactant/nonionic polymer interactions, and the mechanisms responsible for the observed intermicellar exchanges are discussed. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Preparation, Characterization, and Catalytic Properties of Polymer-Stabilized Ruthenium Colloids.

Poly(N-vinyl-2-pyrrolidone) (PVP)-stabilized ruthenium colloids were prepared by NaBH4 reduction at room temperature and characterized by transmission electron microscopy and x-ray photoelectron spectra. The average diameters of the colloids were 1.3-1.8 nm with final sigma = 0.4-0.7 nm with respect to the preparation conditions. The PVP-stabilized ruthenium colloids were used as catalysts in the hydrogenations of cyclooctene and n-heptene and the selective hydrogenation of citronellal to citronellol. Copyright 1998 Academic Press.

Journal Article↗