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Encapsulation of Inorganic Particles by Dispersion Polymerization in Polar Media.

Following a previous work (Bourgeat-Lami, E., and Lang, J., J. Colloid Interface Sci. 197, 293 (1998)), encapsulation of silica beads has been achieved by dispersion polymerization of styrene in an aqueous ethanol medium using poly(N-vinyl pyrrolidone) as stabilizer. Silica beads, prepared according to the Stöber method, were coated prior to polymerization by grafting 3-(trimethoxysilyl)propyl methacrylate onto the surface. A great number of silica beads per composite particle were previously found using beads that had diameters between 49 and 120 nm. In the present work, larger silica beads with diameters between 191 and 629 nm are investigated. We demonstrate by transmission electron microscopy that, consequently, only a small number of silica beads are contained in the composite particles. By counting the composite particles containing precisely zero, one, two, three, four, and more than four silica beads, it clearly appears that the encapsulation of only one silica bead can be obtained simply by increasing the size of the beads. Under our experimental conditions, the optimal bead diameter for achieving composite particles containing only one silica bead turns out to be around 450 nm. We show that increasing the silica bead size above this value results in an increased number of composite particles without silica beads. In contrast, the number of composite particles with two, three, four, or more than four silica beads increases with decreasing silica bead size. In addition to the above variations in composition of the composite particles, changes in particle shapes were also observed as a function of the size of the silica beads and the styrene concentration in the polymerization medium. Hypotheses concerning these variations are presented. Copyright 1999 Academic Press.

Journal Article↗

Preparation and Characterization of Polymer-Stabilized Ruthenium-Platinum and Ruthenium-Palladium Bimetallic Colloids and Their Catalytic Properties for Hydrogenation of o-Chloronitrobenzene.

Colloidal dispersions of poly(N-vinyl-2-pyrrolidone) (PVP)-stabilized ruthenium-platinum and ruthenium-palladium bimetallic colloids were prepared by NaBH4 reduction of the corresponding mixed-metal salts at room temperature and characterized by TEM, XPS, and XRD. The resulting bimetallic colloids were used as catalysts for the selective hydrogenation of o-chloronitrobenzene (o-CNB) in methanol at 303 K under 0.1 MPa of hydrogen. It was observed that the catalytic performance of PVP-stabilized ruthenium-platinum colloids (PVP-Ru/Pt) and ruthenium-palladium colloids (PVP-Ru/Pd) was dependent on their compositions and could be remarkably affected by some added metal cations. In the presence of cobalt ion, nearly 100% selectivity to o-chloroaniline (o-CAN) was achieved over PVP-Ru/Pt colloids at 100% conversion of o-CNB, with an activity two orders of magnitude higher than that of monometallic PVP-Ru colloid. Copyright 1999 Academic Press.

Journal Article↗

Effect of Grafted Amphiphilic PVP-Palmityl Polymers on the Thermotropic Phase Behavior of 1,2 Dipalmitoyl- sn-glycero-3-phosphocholine Bilayer.

To better understand how grafted polymers interact with liposome membrane, a comparative study was conducted to investigate the influence of different chain length polyvinyl pyrrolidone-palmityl (PVP-p) conjugates on the thermotropic phase behavior of 1,2 dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayer. Lipid-polymer dispersions were prepared by mixing DPPC and variable concentrations of PVP-p conjugates in chloroform. Hydration of lipids was performed at 50-55 degrees C after complete elimination of the organic solvent. Differential scanning calorimetry (DSC) was used to determine lipid miscibility and bilayer-polymer interactions. Particle size was determined by photon correlation spectroscopy. Increasing concentrations of 6 kDa PVP-p caused a shift of the main phase transition of DPPC at lower temperatures. At 9.1 mol% the DPPC phase pretransition (T(p)) is abolished. At 16.7 mol%, differential scanning calorimetry showed an endothermic phase transition at 24.9 degrees C. The enthalpy of this transition was twice as high compared to the main phase transition enthalpy of pure DPPC. Inclusion of more than 20 mol% of 6 kDa PVP-p resulted in a complete bilayer micellization. Qualitatively similar to the 6 kDa were the results obtained with the 12 kDa PVP-p conjugate. Increasing concentrations of 25 kDa PVP-p from 1 to 13 mol% resulted in a decrease of the main DPPC phase transition temperature. At 13 mol% the new molecular self-assembled structure as previously identified with the lower MW PVP-p conjugates also showed up at the DSC thermogram. However, in sharp contrast to the lower MW PVP-p conjugates, increasing the 25 kDa PVP-p content did not result in bilayer disruption; rather, it resulted in a bilayer stabilization. The consequences of the hydrophobically modified PVP interaction with the bilayer are considered negative with respect to the long-circulating properties of liposomes in the blood. Copyright 1999 Academic Press.

Journal Article↗

An Aqueous Polymer Two-Phase System as Carrier in the Spray-Drying of Biological Material.

This investigation describes a novel concept in the formulation of carrier systems for the spray-drying of biological materials. As carrier material a system composed of poly(vinyl pyrrolidone) (PVP) and dextran was used. This system yields an aqueous two-phase system in which each phase is enriched in one of the polymers. By varying the composition of the system, the effective structure of a "stirred" system can be varied, covering the entire range from dextran continuous to PVP continuous. This facilitates encapsulation of either of these polymers in a spray-drying operation. In an attempt to investigate the spray-drying from such a system, the surface composition of the spray-dried powder obtained from various compositions of the two-phase system was analyzed by electron spectroscopy for chemical analysis (ESCA), providing information on the distribution of the polymers in the powder and thus also in the spray droplets. The two-phase system was applied for the spray-drying of live bacteria. The survival rate of the bacteria depended on the composition of the two-phase system. The storage stability of the bacteria in these formulations was investigated after storage at room temperature under dry conditions for 4 weeks, and it was found that the survival rate was 10-45%. The results therefore show that this type of formulation holds promise for future applications for micro-organisms as well as other sensitive biological materials such as proteins. Copyright 2000 Academic Press.

Journal Article↗

Protein Adsorption onto Zirconia Modified with Terminally Grafted Polyvinylpyrrolidone.

The potential effectiveness of poly(vinyl pyrrolidone) (PVP) as a zirconia surface modifier for protein adsorption reduction was investigated using lysozyme (LYS). The relatively small size of LYS (45 x 30 x 30 Å) allowed for testing the adequacy of the graft polymerization method for producing a dense surface chain coverage to exclude LYS from direct interaction with the zirconia surface. The study demonstrated that a PVP brush layer is capable of reducing lysozyme adsorption. Overall, the maximum adsorption capacity decreased (by up to about 76%) due to surface modification with increasing polymer/silane surface coverage ratio (mol/mol). Adsorption reduction, due to protein exclusion from the surface by the tethered polymer layer, increased significantly when the distance between surface chains was less than the large axis of LYS (i.e., 45 Å). The present results are encouraging and suggest further consideration of polymer-modified ceramic surfaces for reducing fouling of ceramic membranes during protein ultrafiltration and producing ceramic biocompatible surfaces for biomedical applications. Copyright 2001 Academic Press.

Journal Article↗

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Acetylation↗

Improved preservation of the subepidermal extracellular matrix in axolotl embryos using electron microscopical techniques based on cryoimmobilization.

The purpose of this metholdological survey was to find optimal methods for the fixation and demonstration of glycosaminoglycans, mainly hyaluronan, and proteoglycans, in subepidermal extracellular matrix (ECM) regions of axolotl embryos. We compared living ECM in the laser-scanning microscope (LSM) with chemically fixed or cryoimmobilized extracellular matrix in the transmission (TEM) and scanning electron microscope (SEM). The gel-like structure of living extracellular matrix in the LSM undoubtedly provides the most natural state, whereas shrinkage of the extracellular matrix occurs during conventional fixation and dehydration for TEM or SEM. Among the methods used for fixation and processing of subepidermal extracellular matrices for SEM, plunge-freezing/freeze-drying is to be preferred. Still more satisfying, however, are results obtained with high-pressure frozen/freeze-substituted ECM material in the TEM, for which 10% polyvinyl pyrrolidon +7% methanol was used as a cryoprotectant before high-pressure freezing. In these specimens, no freeze-damage could be observed and they could be regarded as adequately frozen. Conversely, the yield in adequately frozen specimens without cryoprotection was insufficient. In these specimens, the ECM contained honeycomb-like structures which, in the current literature, are regarded as hyaluronan.

Ambystoma↗

Metadoxine prevents damage produced by ethanol and acetaldehyde in hepatocyte and hepatic stellate cells in culture.

Metadoxine (pyridoxine-pyrrolidone carboxylate) has been reported to improve liver function tests in alcoholic patients. In the present work we have investigated the effect of metadoxine on some parameters of cellular damage in hepatocytes and hepatic stellate cells in culture treated with ethanol and acetaldehyde. HepG2 and CFSC-2G cells were treated with 50 mM ethanol or 175 microM acetaldehyde as initial concentration in the presence or absence of 10 microg ml(-1) of metadoxine. Twenty-four hours later reduced and oxidized glutathione content, lipid peroxidation damage, collagen secretion and IL-6, IL-8 and TNF- alpha secretion were determined. Our results suggest that metadoxine prevents glutathione depletion and the increase in lipid peroxidation damage caused by ethanol and acetaldehyde in HepG2 cells. In hepatic stellate cells, metadoxine prevents the increase in collagen and attenuated TNF- alpha secretion caused by acetaldehyde. Thus, metadoxine could be useful in preventing the damage produced in early stages of alcoholic liver disease as it prevents the redox imbalance of the hepatocytes and prevents TNF- alpha induction, one of the earliest events in hepatic damage.

Acetaldehyde↗

N-methyl-D-aspartate receptor antagonists counteract the long lasting 5-HT1A receptor-induced attenuation of postsynaptic responses in the rat in vivo.

The effect of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists dizocilpine and phenycyclidine, the competitive NMDA antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) and the antagonist at the glycine modulatory site of the NMDA receptor, 3-amino-1-hydroxy-2-pyrrolidone (HA-966) on the long lasting attenuation of some post-synaptic 5-HT1A receptor-mediated responses in rats (increased corticosterone secretion and inhibition of the cage leaving response) produced by a single injection of the 5-hydroxytryptamine1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was studied. It was found that these antagonists counteracted the attenuation of these responses at dose levels known to block the NMDA receptor-ion channel complex in vivo. It is concluded that the long lasting attenuation of postsynaptic responses after a 5-HT1A receptor agonist is initiated through stimulation of glutamate NMDA receptors indicating a functional interaction between the 5-HT and glutamate systems in at least two different models.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

Cerebral-activating (EEG) properties of two inverse agonists and of an antagonist at the benzodiazepine receptor in the rat.

In order to assess the effects of inverse benzodiazepine agonists and antagonists on brain function, computerized EEG (CEEG) analysis was performed in rats following the i.p. administration of SR 95195 (7-phenyl-3-methyl-1,2,4 triazolo-[4,3-b]pyridazine) and CGS 8216 (2-phenylpyrazolo-[4,3c]-quinoline-3-[5H]-one) two benzodiazepine receptor inverse agonists (BRIAGs) and of flumazepil (Ro 15-1788), a benzodiazepine receptor antagonist (BRANT). The EEG effects of SR 95195 (3, 10, 30 and 60 mg/kg), CGS 8216 (10 and 30 mg/kg) and flumazepil (3, 10, 30 and 60 mg/kg) were compared to those of the psychostimulant drugs DL-amphetamine (0.1, 0.3 and 1 mg/kg), and caffeine (10 and 30 mg/kg) and those of aniracetam (100 and 300 mg/kg), a nootropic pyrrolidone derivative. The CEEG profiles of SR 95195, CGS 8216 and flumazepil were mainly characterized by a power increase in the 20-32 Hz frequency range and by a power reduction in the 8-16 Hz range. These effects were quite similar to those of the psychostimulants DL-amphetamine and caffeine as well as to those of the nootropic aniracetam. Other psychotropic drugs with CNS-depressant properties, namely diazepam (10 mg/kg p.o.), pentobarbital (30 mg/kg p.o.), chlorpromazine (10 mg/kg i.p.) and imipramine (10 mg/kg i.p.) induced quite different EEG power modifications. These results show that BRIAGs and BRANTs possess a marked intrinsic activity at the central level and suggest that this activity is CNS-activating in nature.

Animals↗

Effects of forskolin and cyclic nucleotides in animal models predictive of antidepressant activity: interactions with rolipram.

Forskolin, a direct activator of the catalytic subunit of adenylate cyclase (AC), and the cyclic nucleotide analogs dibutyryl cAMP (dBcAMP), 8-bromo cAMP (8-BrcAMP) and dibutyryl cGMP (dBcGMP) were tested for their ability to reverse the hypothermia or hypokinesia of mice depleted of presynaptic endogenous monoamines by pretreatment with reserpine, alpha-methyl-p-tyrosine and p-chlorophenylalanine. Forskolin and the cAMP analogs decreased the rectal temperature and inhibited locomotor activity in normal mice. In mice depleted of brain monoamines forskolin reversed the hypothermia and hypokinesia; dBcAMP and 8-BrcAMP antagonized the hypothermia but were only marginally effective in reversing the hypokinesia. DBcGMP was inactive. The antihypothermic action of forskolin or salbutamol was enhanced by the novel antidepressant and cAMP selective phosphodiesterase inhibitor rolipram (4RS-[3-cyclopentyloxy-4-methoxy-phenyl]-2-pyrrolidone). As an indirect effect via release of endogenous monoamines stimulating postsynaptic receptors was precluded by the monoamine-depleting pretreatment, forskolin and the cAMP analogs are thought to exert their antidepressant action by directly increasing brain cAMP availability. This is achieved by forskolin via activation of the catalytic subunit of AC and by the cAMP analogs via substitution for cAMP. These findings suggest that antidepressant activity is crucially linked to enhanced cAMP availability within brain effector cells. The successful treatment of endogenously depressed patients with rolipram supports this assumption.

8-Bromo Cyclic Adenosine Monophosphate↗

Behavioral and physiological effects associated with changes in muscarinic receptors following administration of an irreversible cholinergic agonist (BM 123).

Previous work in our laboratory has shown that the aziridinium ion of BM 123 (N-[4(2-chloroethylmethylamino)-2-butynyl]-2 pyrrolidone) is a potent and selective muscarinic agonist and binds irreversibly to muscarinic receptors (mAChR). The present series of experiments was designed to study the effects of BM 123 on behavioral and physiological variables known to be sensitive to manipulations of the cholinergic neurotransmitter system. BM 123 was injected into the tail vein of Sprague-Dawley rats, reducing mAChR to approximately 10% of normal as judged by [3H](-)QNB binding. Oxotremorine was injected IV for purposes of comparison. Behavioral and physiological variables were measured daily for 26 days. Physiological variables (e.g., tremor, chromodacryorrhea, salivation, and temperature) showed effects in less than 5 min after injection and returned to their pretreatment baselines within minutes. Nociceptive thresholds, dependent on sensory-perceptual processes, showed peak changes of approximately +230% and returned to normal within hours. Motoric responses, i.e., drinking and general activity, recovered in 3-4 days. Learned responses and those requiring temporal discrimination took 8-11 days to recover and were the only responses paralleling the return of the mAChRs to their normal levels. Changes elicited by oxotremorine recovered more rapidly than those elicited by BM 123. The results suggest that the different variables measured are dependent on different densities of functional receptors. Implications for a theoretical model are discussed.

Animals↗

Pyroglutamic acid. Non-metabolic formation, function in proteins and peptides, and characteristics of the enzymes effecting its removal.

The formation of pyrrolidone carboxylic acid (PCA, pGlu) during protein biosynthesis is discussed. Studies are summarized which demonstrate that PCA is formed during the later stages of biosynthesis at the terminal phases of translation or as a post-translational event, just prior to cellular secretion of protein with amino-terminal PCA. Of the studies cited, the most convincing evidence suggests that PCA is derived from glutamine. Enzymes which selectivity remove PCA from the N-terminus, and of benefit in amino-acid sequence analysis, have been isolated and shown to have a ubiquitous distribution in various animal and plant cells. The investigations which lead to the isolation o these enzymes and the procedures for their use in removing amino-terminal PCA from proteins, are described. Finally, the biologic function of PCA and the effects of its chemical modification are discussed using the neuropeptide. Thyrotropin Releasing Factor (TRF) as a specific example.

Amino Acid Sequence↗

Lactam utilisation in Aspergillus nidulans: evidence for a fourth gene under the control of the integrator gene intA.

Mutations in the lamA gene of Aspergillus nidulans prevent the conversion of exogenous 2-pyrrolidone (gamma-butyrolactam) to gamma-amino-n-butyrate (GABA) and also prevent the (probably analogous) utilisation of 2-piperidone (sigma-valerolactam). The lamA gene, in linkage group VIII, probably specifies a lactamase but a rôle in lactam uptake cannot be ruled out. lamA is probably under the control of the positive acting regulatory gene intA, which can integrate its expression with the expression of three other genes, specifying acetamidase, GABA transaminase, and GABA permease, respectively.

Aspergillus nidulans↗

In-patient major depression: is rolipram as effective as amitriptyline?

The antidepressant efficacy and adverse-effects of rolipram (a dialkoxyphenyl-2-pyrrolidone) were compared to those of amitriptyline in the treatment of depressive illness requiring hospital admission in a double-blind study. Fifty patients meeting DSM-III criteria for Major Depression whose scores on the Hamilton Rating Scale for Depression (HRSD) remained above 17 after 5 to 7 days on placebo were randomly allocated to either treatment. The rate of recovery in those patients treated by amitriptyline was substantially greater than in those patients treated by rolipram. Twice as many patients dropped out of treatment by rolipram because of lack of efficacy or adverse-effects compared with patients treated by amitriptyline. Rolipram produced fewer adverse-effects attributable to cholinergic blockade, but more nausea. We conclude that amitriptyline is more effective than rolipram in the treatment of depressed hospital in-patients.

Adolescent↗

Alveolitis due to hair-spray. Ultrastructural observations in two patients and the results of experimental investigations.

Observation of two patients with hair-spray induced lung disease have prompted us to study the ultrastructure of the lung lesion. We have compared the results with experimental lesions in animals injected with hair-spray extracts and with human monocyte cell cultures exposed to hair-spray. The lungs show a chronic alveolitis with a striking granulomatous reaction including macrophages and multinucleated giant cells of the foreign body type. The intraalveolar and interstitial macrophages and the giant cells all contain PAS-positive material. Ultrastructurally distinct lamellar inclusions are found in the secondary lysosomes of the macrophages and giant cells. Identical structures can be produced in animals injected with hair-spray extracts and with polyvinyl-pyrrolidone and -acetate (PVP/PVA), which are regular ingredients of hair-sprays. Large, presumeably polymeric particles (PVP/PVA) are ingested by giant cells. This "gigantophagocytosis" is associated with the fusion of mononuclear phagocytes and leads to the genesis of giant cells. In cell cultures of human blood monocytes hair-spray extracts and PVP/PVA induce maturation and aggregation of these cells, with PAS-positive cytoplasmatic inclusions. The development of multinuclear giant cells in these monocyte cell cultures is also seen. These observations suggest that hair-spray induced lung disease is caused by the prolonged and extensive body response of the local mononuclear phagocyte system (MPS). Overstimulation of the MPS leads to a quantitative and qualitative change which is followed by a partial blockade of this system. The alveolitis is a consequence of the foreign body response to inhaled hair-spray substances.

Female↗

Attenuation of delta 9-tetrahydrocannabinol-induced withdrawal-like behaviour by delta 9-tetrahydrocannabinol.

Rats chronically treated with increasing daily doses of delta 9-tetrahydrocannabinol (delta 9-THC) for up to 5 or 10 days exhibit a withdrawal-like behavioural response when challenged with clomipramine, a potent inhibitor of serotonin (5-hydroxytryptamine) reuptake, at the time when the next injection of delta 9-THC was due. To determine whether this response is indeed a withdrawal syndrome, different groups of rats were injected IV twice daily for up to 5 days with either delta 9-THC, in doses increasing from 2-6 mg/kg, or polyvinyl-pyrrolidone (PVP), the vehicle in which delta 9-THC was suspended. On day 6, an acute dose of 6 mg/kg delta 9-THC was given 30 min before IP clomipramine (15 mg/kg). The total behavioural response, which included writhing, backward kicking, jumping, head shaking, and paw tremor, was attenuated in rats chronically treated with delta 9-THC, but not in rats which received an acute dose of PVP. These results lend further evidence to our hypothesis that chronically administered delta 9-THC produces a state of physical dependence in the rat.

Animals↗

Effect of stabilizers on diaminobenzidine reactions of mitochondria.

The effect of various stabilizers--0.9% NaCl, 7.5% sucrose and 7.5% polyvinyl pyrrolidone (PVP) on reactions of mitochondria with fresh and photooxidized diaminobenzidine (DAB) was investigated. NaCl and PVP abolished the DAB staining of mitochondria incubated without exogenous cytochrome c; NaCl, however, was ineffective when exogenous cytochrome c or oxidized DAB were present in the incubation medium. PVP considerably decreased the intensity of all reactions performed in the presence of exogenous cytochrome c or oxidized DAB. The mechanism of the observed effect is not likely to involve osmotic protection of mitochondria; extraction of endogenous cytochrome c from fresh frozen tissue by NaCl and greatly increased viscosity of PVP-containing media seem to be most probable explanations.

3,3'-Diaminobenzidine↗