Adsorption effectiveness of ß-lactoglobulin onto gold surface determined by quartz crystal microbalance.

Bovine ß-lactoglobulin (LGB) is a transport protein that can bind to its structure hydrophobic bioactive molecules. Due to the lack of toxicity, high stability and pH-dependent molecular binding mechanism, lactoglobulin can be used as a carrier of sparingly soluble drugs. Dynamic light scattering has confirmed LGB's tendency to create oligomeric forms. The hydrodynamic diameter of LGB molecules varies from 4 nm to 6 nm in the pH range of 2-10 and ionic strength I = 0.001-0.15 M, which corresponds to the presence of mono or dimeric LGB forms. The LGB zeta potential varies from 26.5 mV to -33.3 mV for I = 0.01 M and from 13.3 mV to -16 mV for I = 0.15 M in the pH range of 2-10. The isoelectric point is at pH 4.8. As a result of strong surface charge compensation, the maximum effective ionization degree of the LGB molecule is 35% for ionic strength I = 0.01 M and 22% for I = 0.15 M. The effectiveness of adsorption is linked with the properties of the protein, as well as those of the adsorption surface. The functionalization of gold surfaces with ß-lactoglobulin (LGB) was studied using a quartz crystal microbalance with energy dissipation monitoring (QCM-D). The effectiveness of LGB adsorption correlates strongly with a charge of gold surface and the zeta potential of the molecule. The greatest value of the adsorbed mass was observed in the pH range in which LGB has a positive zeta potential values, below pH 4.8. This observation shows that electrostatic interactions play a dominant role in LGB adsorption on gold surfaces. Based on the adsorbed mass, protein orientation on gold surfaces was determined. The preferential side-on orientation of LGB molecules observed in the adsorption layer is consistent with the direction of the molecule dipole momentum determined by molecular dynamics simulations of the protein (MD). The use of the QCM-D method also allowed us to determine the effectiveness of adsorption of LGB on gold surface. Knowing the mechanism of LGB adsorption is significant importance for determining the optimum conditions for immobilizing this protein on solid surfaces. As ß-lactoglobulin is a protein that binds various ligands, the binding properties of immobilized ß-lactoglobulin can be used to design controlled protein structures for biomedical applications.

Investigation of high pressure effect on the structure and adsorption of ß-lactoglobulin.

ß-Lactoglobulin, being one of the principal whey protein, is of huge importance to the food industry. Temperature/pressure effects on this small protein has been extensively studied by industry. To characterize biochemical properties of ß-lactoglobulin after or during pressurization, a wide range of methods have been used thus far. In this study, for the first time, the pressure-induced conformation of ß-lactoglobulin in the crystal state was determined, at pressure 430 MPa. Changes observed in the high pressure structure correlate with the physico-chemical properties of pressure-treated ß-lactoglobulin obtained from dynamic light scattering, electrophoretic mobility and quartz crystal microbalance with dissipation monitoring measurements. A comparison between the ß-lactoglobulin structures determined at both high and ambient pressure contrasts the stable nature of the protein core and adjacent loop fragments. At high pressure the ß-lactoglobulin structure presents early signs of dimer dissociation, charge and conformational changes characteristic for initial unfolded intermediate as well as a significant modification of the binding pocket volume. Those observations are supported by changes in zeta potential values and results in increase affinity of the ß-lactoglobulin adsorption onto gold surface. Observed pressure-induced structural modifications were previously suggested as an important factor contributing to ß-lactoglobulin denaturation process. Presented studies provide detailed analysis of pressure-associated structural changes influencing ß-lactoglobulin conformation and consequently its adsorption.

Urachal Adenocarcinoma in a Dog.

An 8-year-old neutered female Labrador retriever was presented with a 3-year history of intermittent haematuria. Ultrasonographic evaluation of the urinary bladder revealed a 2 × 3 × 0.5 cm intraluminal mass arising at the dome. The mass was excised via partial cystectomy. Histopathological examination revealed neoplastic epithelial cells arranged in sheets, irregularly-branching tubules and acini within a fibrovascular stroma. Neoplastic cells were cuboidal to polygonal with abundant foamy amphophilic cytoplasm, typically with a single, large, clear intracytoplasmic vacuole and eccentric nucleus ('signet ring' cells). Neoplastic tubules were often ectatic and contained abundant mucin. Immunohistochemically, the neoplastic cells had weak, cytoplasmic immunoreactivity for cytokeratin 7 and rare, but strong, nuclear immunoreactivity for CDX2. Based on the cellular morphology, immunolabelling characteristics and anatomical location, a diagnosis of adenocarcinoma of urachal origin was made. To the authors' knowledge, this is the first reported case of urachal adenocarcinoma in a dog.

Evaporation as the transport mechanism of metals in arid regions.

Soils of arid regions are exposed to drought and drastic temperature oscillations throughout the year. Transport mechanisms in these soils are therefore very different from the ones in temperate regions, where rain dictates the fate of most elements in soils. Due to the low rainfall and high evaporation rates in arid regions, groundwater quality is not threatened and all soil contamination issues tend to be overlooked. But if soil contamination happens, where do contaminants go? This study tests the hypothesis of upward metal movement in soils when evaporation is the main transport mechanism. Laboratory evaporation tests were carried out with heavy metal spiked Saudi soil, using circulation of air as the driving force (Fig. 1). Main results show that loamy soil retains heavy metals quite well while evaporation drives heavy metals to the surface of a sandy soil. Evaporation transports heavy metals upward in sandy soils of arid regions, making them accumulate at the soil surface. Sand being the dominating type of soil in arid regions, soils can then be a potential source of contaminated aerosols and atmospheric pollution - a transboundary problem. Some other repercussions for this problem are foreseen, such as the public ingestion or inhalation of dust.

Assessing PAH removal from clayey soil by means of electro-osmosis and electrodialysis.

Polycyclic aromatic hydrocarbons (PAH) are persistent and toxic contaminants which are difficult to remove from fine porous material like clayey soils. The present work aims at studying two electroremediation techniques for the removal of PAHs from a spiked natural silt soil from Saudi Arabia and a silty loam soil from The Netherlands which has been exposed to tar contamination for over 100 years. The two techniques at focus are electro-osmosis and electrodialysis. The latter is applied for the first time for the removal of PAH. The efficiency of the techniques is studied using these two soils, having been subjected to different PAH contact times. Two surfactants were used: the non-ionic surfactant Tween 80 and anionic surfactant sodium dodecyl sulphate (SDS) to aid desorption of PAHs from the soil. Results show a large discrepancy in the removal rates between spiked soil and long-term field contaminated soil, as expected. In spiked soil, electro-osmosis achieves up to 85% while electrodialysis accomplishes 68% PAH removal. In field contaminated soil, electro-osmosis results in 35% PAH removal whereas electrodialysis results in 79%. Short recommendations are derived for the up-scale of the two techniques.

[Pneumomediastinum, giant subcutaneous emphysema and pneumoperitoneum revealed by jaw pain. Uncommon physiopathology of pneumomediastinum]. / Pneumomédiastin, emphysème sous-cutané géant et pneumopéritoine d'origine dentaire Physiopathologie inhabituelle d'un pneumomédiastin.

INTRODUCTION: Spontaneous pneumomediastinum is a rare entity, predominantly described in young man. The association of acute dyspnea, chest pains and subcutaneous emphysema is usually reported. CASE REPORT: We report the observation of a pneumomediastinum, fortuitously discovered in front of an isolated giant subcutaneous emphysema in a 59 year old man. The recent clinical history was only marked by the presence of intense and acute dental pains. Associated with a pneumoperitoneum, a retro-pneumoperitoneum, this clinical presentation is uncommon and differs from previous published case reports. Despite a complete evaluation of classical risk factors, its origin remains uncertain. However, the presence of huge dental injuries led to consider such local origin, facilitating air diffusion. CONCLUSION: This case report allows to reconsider spontaneous pneumomediastinum entity and to propose additional physiopathological mechanisms. This original description underlines the interest to systematically perform dental examination in the presence of unexplained pneumomediastinum.

[Aspergillus bronchitis and aspergilloma treated successfully with voriconazole]. / Un cas d'aspergillome associé à une bronchite aspergillaire traités par Voriconazole.

INTRODUCTION: Aspergillus fumigatus is a ubiquitous soil-dwelling organism, which can cause both aspergillomas which develop in a preformed lung cavity, and aspergillus bronchitis. The two pathologies can occasionally co-exist, notably in patients with of cystic fibrosis. CASE REPORT: We describe a 57 year old patient, with diffuse bronchiectasis, who developed aspergillus bronchitis as well as an aspergilloma complicating a cavity caused by an atypical mycobacterial infection. After one month of therapy with voriconazole the aspergilloma had decreased in size and the endobronchial changes had resolved. CONCLUSION: This case report illustrates that in addition to its established role for the treatment of invasive aspergillosis, voriconazole is a promising new therapy for the treatment of aspergilloma and aspergillus bronchitis.

Quantifying the effect of membrane potential in chemical osmosis across bentonite membranes by virtual short-circuiting.

Clay liners are charged membranes and show semipermeable behavior regarding the flow of fluids, electrical charge, chemicals and heat. At zero gradients of temperature and hydrostatic pressure, a salt concentration gradient across a compacted clay sample induces not only an osmotic flux of water and diffusion of salt across the membrane but also an electrical potential gradient, defined as membrane potential. Laboratory experiments were performed on commercially available bentonite samples in a rigid-wall permeameter connected to two electrically insulated fluid reservoirs filled with NaCl solutions of different concentrations and equipped with Ag/AgCl electrodes to measure the electrical potential gradient. The effect of membrane potential could be cancelled out by short-circuiting the clay with the so-called virtual shortcut. The potential gradient across the sample is brought to zero with a negative feedback circuit. It was observed that the water flux and the diffusion of Cl- were hindered by the occurrence of a membrane potential, indicating that an electroosmotic counterflow is induced. Flow parameters were calculated with modified coupled flow equations of irreversible thermodynamics. They were in excellent agreement with values reported in the literature. Comparing the method of short-circuiting with a study elsewhere, where the electrodes were physically short-circuited, it was shown that the virtual shortcut is more appropriate because physically short-circuiting induces additional effects that are attributed to the fluxes.

Dissolution kinetics of heavy metals in Dutch carbonate- and sulfide-rich freshwater sediments.

In two sulfide-rich freshwater sediments from the Biesbosch and Kromme Rijn River in the Netherlands differing in carbonate content and acid volatile sulfide (AVS) content, metal and sulfide dissolution kinetics were studied at different acid concentrations by varying both the procedure of acid addition and the extraction time. The establishment of equilibrium was monitored by measuring the pH in time, which reached a near constant value. The equilibrium pH was reached quickly when large amounts of acid were added and slowly when small amounts of acid were added. This observation was confirmed by the yield of extracted metals after either a 45-min or 24-h extraction over a pH range from 0 to 5. The pH factor seemed to be of more influence than time for the dissolution of metals. The amount of extracted metals was highly dependent on the metal itself due to its physico-chemical behavior. Although the sediments studied varied in carbonate content, acid volatile sulfide (AVS), and total metal content, the extracted fraction of metals compared with their total content in the sediment was similar for most metals. Finally, the AVS content as well as the ratio of simultaneously extracted metals (SEM; sum of Cd, Cu, Ni, Pb, and Zn) to AVS decreased with increasing pH. Because the SEM to AVS ratio may be used to set environmental quality criteria for the sediment compartment, this observation is of significance.

Computed ligand electronic parameters from quantum chemistry and their relation to Tolman parameters, Lever parameters, and Hammett constants.

The calculated (DFT, B3PW91) A(1) nu(CO) frequency in LNi(CO)(3) defines an electronic parameter that reliably predicts the relative donor powers of a wide variety of cationic, neutral, and negatively charged ligands. These calculated parameters correlate very well with the available Tolman and Lever parameters, and also with Hammett's sigma(m), where available. The method avoids any experimental limitations and, in particular, can be used for proposed ligands not yet experimentally available.

The 5-HT3 antagonist tropisetron (ICS 205-930) is a potent and selective alpha7 nicotinic receptor partial agonist.

The 5-HT3 receptor antagonist tropisetron (ICS 205-930) was found to be a potent and selective partial agonist at alpha7 nicotinic receptors. Two other 5-HT3 receptor antagonists, ondansetron and LY-278,584, were found to lack high affinity at the alpha7 nicotinic receptor. Quinuclidine analogues (1 and 2) of tropisetron were also found to be potent and selective partial agonists at alpha7 nicotinic receptors.

(-)-Spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin-2'-one], a conformationally restricted analogue of acetylcholine, is a highly selective full agonist at the alpha 7 nicotinic acetylcholine receptor.

Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel receptor superfamily and may play important roles in modulating neurotransmission, cognition, sensory gating, and anxiety. Because of its distribution and abundance in the CNS, the alpha 7 nicotinic receptor is a strong candidate to be involved in some of these functions. In this paper we describe the synthesis and in vitro profile of AR-R17779, (-)-spiro[1-azabicyclo[2.2. 2]octane-3,5'-oxazolidin-2'-one] (4a), a potent full agonist at the rat alpha 7 nicotinic receptor, which is highly selective for the rat alpha 7 nicotinic receptor over the alpha 4 beta 2 subtype. Preliminary SAR of AR-R17779 presented here indicate that there is little scope for modification of this rigid molecule as even minor changes result in significant loss of the alpha 7 nicotinic receptor affinity.

The effect of oxidation and acidification on the speciation of heavy metals in sulfide-rich freshwater sediments using a sequential extraction procedure.

The speciation of metals in a contaminated, anoxic, sulfide-rich, freshwater sediment was determined experimentally, using a sequential extraction procedure based on the method of Tessier et al. Taking into account the advantages and disadvantages of sequential extractions, the applied methodology allowed the investigation of the influence of aeration and acidification on the distribution of various metals in the sediment. Aeration caused Zn and Cd to be released from sulfides. Carbonates were partly dissolved by the oxidation process, causing mobilisation of Ca. Fe became less mobile owing to a stronger binding to organic matter. The speciation of K, Al, Ni, Pb and Mn and to a lesser extent of Cu was not affected by aeration. As a result of acidification of the aerated sediment, Ca, Mn, Ni, Zn and Cd became more mobile owing to the dissolution of carbonates.

Determination of antibody responses of elderly adults to all 23 capsular polysaccharides after pneumococcal vaccination.

The 23-valent pneumococcal polysaccharide vaccine was formulated to prevent invasive infection in the elderly and other high-risk populations from the most prevalent Streptococcus pneumoniae serotypes. However, the immunogenicity of all 23 vaccine polysaccharides has not been fully characterized in elderly adults. We previously reported that whereas the majority of elderly subjects had vigorous immune responses to selected pneumococcal vaccine polysaccharides, a subset of elderly individuals responded to fewer than two of seven vaccine serotypes after immunization. To determine whether these elderly low responders have a general inability to respond to pneumococcal vaccine and to determine whether elderly low responders might be identified by their responses to a few polysaccharides, we measured antibody responses of elderly adults to all 23 vaccine polysaccharides after pneumococcal immunization. As a group, elderly subjects showed a significant rise after immunization in geometric mean antibody levels to all 23 vaccine serotypes. However, when individual rather than group immune responses were assessed, the 23-valent vaccine did not appear to be uniformly immunogenic in these elderly subjects. Eleven elderly subjects (20%) had twofold increases in specific antibody after vaccination to only 5 or fewer of the 23 vaccine polysaccharides, and they did not respond to the most prevalent serotypes causing invasive disease. Antibody responses to serotype 9N were found to reliably distinguish low vaccine responders from other elderly subjects. However, no particular group of vaccine polysaccharides could be used as a marker for adequate immune responses if only postvaccination sera were analyzed.

Magnitude, duration, quality, and function of pneumococcal vaccine responses in elderly adults.

The suboptimal efficacy of the currently available 23-valent pneumococcal vaccine in the growing population of adults >65 years old may be related to the limited immunogenicity of the vaccine polysaccharides in this group. In this study, the majority of elderly outpatients with stable chronic illnesses generated a vigorous IgG response to seven vaccine serotypes comparable to that of healthy young adults at 1, 3, and 16 months after immunization. Moreover, the quality and function of anticapsular antibodies, measured as avidity and in vitro opsonization, were comparable between elderly and young subjects over time. However, a subset (approximately 20%) of elderly outpatients responded to fewer than two of seven serotypes tested 1 and 3 months after immunization, whereas none of the healthy young adults were such poor responders. Thus, despite the adequate mean immune responses of the elderly as a group, a substantial proportion of elderly persons may have poor responses to the currently available pneumococcal vaccine.

In vitro muscarinic activity of spiromuscarones and related analogs.

The cholinergic hypothesis of Alzheimer's disease suggests that cholinergic agonists may have therapeutic potential for treating the attendant memory deficits of the disease. As part of a program aimed at preparing metabolically stable, nonquaternary analogs of muscarone, 1-oxa-2,8-dimethyl-8-azaspiro[4.5]decan-3-one, 2a, and related analogs have been synthesized and their in vitro muscarinic activity evaluated. The synthetic strategy in the formation of the 1-spiro[4.5]decan-3-one ring system of 2a involved cyclization of the diol 4 in the presence of Nafion-Hg. The spiromuscarone 2a was found to displace [3H]Oxo-M binding with a Ki value of 7 nM. Affinities of the oxime and hydrazone analogs of 2a were lower than 2a. The compounds in these series were partial muscarinic agonists as demonstrated by stimulation of phosphatidyl inositol hydrolysis assay, with 2a showing the highest intrinsic intrinsic activity (60% as compared with carbachol). The results from this study indicate that an exo double bond at the C-3 position is essential for the receptor binding.

The influence of anesthetic agents on rat hepatic cytochromes P450 in vivo.

The objective of this study was to identify which anesthetics when used acutely will affect cytochrome P450 (CYP) activity in male Sprague-Dawley rats in vivo. The anesthetics tested were fentanyl citrate, alpha-chloralose, ketamine, urethane (ethyl carbamate), halothane, and ether. CO2 anesthesia was used as the control comparator. Theophylline was used as a probe for CYP1A activity, phenobarbital for CYP2B/2C, flecainide for CYP2D1, and ethosuximide for CYP3A activity. All probes were administered via tail vein injection after anesthetic-induced loss of the righting reflex. Single sample probe clearances were estimated, and used as an index of CYP activity. Fentanyl citrate, alpha-chloralose, halothane, and ether did not have statistically significant effects on any of the CYP activities. Ketamine did not significantly affect CYP1 or CYP2B/2C activity. However, it decreased the clearance of flecainide (i.e. CYP2D1 activity) by 13.4% (p < 0.001) and the clearance of ethosuximide (i.e. CYP3A activity) by 17.6% (p < 0.0001). Urethane increased the clearance of theophylline by 91.5% (p < 0.0001), and decreased the clearance of ethosuximide by 40.5% (p < 0.0001) though it did not affect CYP2B/2C or CYP2D1 activities significantly. From this data, we conclude that a single dose of ketamine mildly inhibits the activity of CYP2D1 and CYP3A, and a single dose of urethane strongly inhibits CYP3A but increases CYP1A activity.

In vivo evidence that theophylline is metabolized principally by CYP1A in rats.

The role of various subfamilies of rat hepatic cytochrome P-450 in the oxidation of theophylline was evaluated by comparing theophylline clearance in control rats and those pretreated with relatively selective inducers and inhibitors of the cytochromes P-450. Pretreatment with the CYP1A inducer, beta-naphthoflavone (BNF), increased theophylline clearance 4.5-fold (p < 0.001), and the CYP1A inhibitor, alpha-naphthoflavone, significantly attenuated the BNF effect. Pretreatment with phenobarbital, an inducer of CYP2B/C in rats, had a far more modest effect, increasing theophylline clearance only 1.6-fold (p < 0.005). The phenobarbital-mediated increase in theophylline clearance was attenuated by orphenadrine, a CYP2B/C inhibitor. The CYP2E inducer, isoniazid and the CYP2E inhibitor, diallyl sulfide were virtually without effect, as was the CYP4A inducer, clofibrate, and the CYP4A inhibitor, 10-undecynoic acid. Ajmaline, and inhibitor of CYP2D, was also without any effect on theophylline clearance. While the powerful CYP3A inducer clotrimazole did not increase theophylline clearance, troleandomycin, an inhibitor of CYP3A, did slow theophylline clearance by about 25% (p < 0.002). Together, these findings suggest that CYP1A is principally responsible for the overall oxidation of theophylline in rats, and that CYP2B/C probably also mediates some theophylline oxidation. The involvement of CYP2D, CYP2E, CYP4A, and CYP3A is relatively trivial.