The blue copper ceruloplasmin induces aggregation of newly differentiated neurons: a potential modulator of nervous system organization.

Ceruloplasmin (CP) is a copper-dependent ferroxidase. It regulates iron metabolism and is involved in inflammation, angiogenesis, and protection against oxidative stress. CP also modulates K(+) channel activity in neuroblastoma cells and affects cardiodynamics of isolated hearts. Considering the presence of CP in the nervous system and the importance of iron ions and K(+) channels in neuronal activity, we postulated a role of CP in neuronal development. This hypothesis was tested using the P19 mouse embryonal carcinoma cell line, a model of neuronal differentiation. Addition of CP to the culture medium of newly differentiated P19 neurons induced cell aggregation within 24 h. This effect was concentration-dependent half-maximal at 50 nM, and not associated with necrosis, apoptosis or changes in secretory function. Deglycosylated CP was aggregative but not denatured CP, copper salts, His(2)Cu complex, or other copper enzymes or serum proteins. CP-induced aggregation was less pronounced with aging neurons and seemed not to involve K(+) channels. Immunocytofluorescence analysis demonstrated that digoxigenin-labeled CP bound to P19 neurons and the proportion of responding neurons decreased with aging. The interaction of digoxigenin-labeled CP with neurons was half-maximal at 120 nM by enzyme-linked immunosorbent assay and displaced by unlabeled CP. Our data indicate a specific aggregative action of CP on young neurons in vitro, possibly involving CP receptors. A potential developmental role of CP in nervous system organization is thus demonstrated.

Novel cardiac protective effects of urea: from shark to rat.

1. This study was carried out to investigate novel cardioprotective effects of urea and the underlying mechanisms. The cardiac functions under oxidative stress were evaluated using Langendorff perfused isolated heart. 2. Isolated dogfish shark hearts tolerated the oxidative stress generated by electrolysis (10 mA, 1 min) of the perfusion solution (n=4), and also showed normal cardiac functions during post-ischaemia reperfusion (n=4). The high concentration of urea (350 mM) in the heart perfusate was indispensable for maintaining the normal cardiac functions of the shark heart. 3. Urea at 3 - 300 mM (n=4 for each group) protected the isolated rat heart against both electrolysis-induced heart damage and post-ischaemia reperfusion-induced cardiac injury. 4. A concentration-dependent scavenging effect of urea (3 - 300 mM, n=4 for each group) against electrolysis-induced reactive oxygen species was also demonstrated in vitro. 5. Urea derivatives as hydroxyurea, dimethylurea, and thiourea had antioxidant cardioprotective effect against the electrolysis-induced cardiac dysfunction of rat heart, but were not as effective as urea in suppressing the post-ischaemia reperfusion injury. 6. Our results suggest that urea and its derivatives are potential antioxidant cardioprotective agents against oxidative stress-induced myocardium damage including the post-ischaemia reperfusion-induced injury.

Cross-linked amylose tablets containing alpha-amylase: an enzymatically-controlled drug release system.

An oral controlled release system based on direct compression of cross-linked amylose (CLA) and drug powders was previously introduced. For drugs with limited solubility or for some drugs for which solubility can be influenced by variation of gastro-intestinal pH, a system is required to accelerate drug release. This paper describes a novel enzymatically-controlled drug release (ECDR) system based on the addition of alpha-amylase to CLA tablets, which can modulate the release kinetics of drugs. The alpha-amylase within the tablets is able to hydrolyze alpha-1-4-glucosidic bonds present in the CLA semisynthetic substrate. Increasing amounts of alpha-amylase (5 to 25 EU) within the tablets induced a significant decrease in release time from 24 to 6 h. High amounts of external alpha-amylase (300-6000 EU/l) had a slight effect on the release rate. Drug release from the ECDR system seems to be controlled by two sequential mechanisms: (a) hydration and swelling of CLA tablets followed by (b) internal enzymatic hydrolysis of the hydrated gel phase.

Cross-linked high amylose starch derivatives as matrices for controlled release of high drug loadings.

Selection of hydrogels as excipients in controlled drug release systems depends on the characteristics of the gel and of the drug. Three types of derivatives were synthesized from cross-linked high amylose starch (HASCL-6) by substitution of hydroxylic groups with cationic (carboxymethyl: CM), anionic (aminoethyl: AE) and acetate (Ac) groups. These new polymeric excipients are able to control the release over 20 h from monolithic tablets loaded with 20 to 60% drug. Three drugs were used as model tracer: acetaminophen (uncharged), acetylsalicylic acid (having an acidic group) and metformin (having a basic group). It was found that the release of ionic drugs from CM-HASCL-6 and AE-HASCL-6 matrices can be partially controlled by ionic interaction between pendant groups of polymer and drugs. The substitution degree of HASCL-6 derivatives can also be varied to modulate the drug's release time. These derivatives represent a novel generation of pharmaceutical excipients, recommended for high loading dosage formulations.

Cross-linked high amylose starch for controlled release of drugs: recent advances.

Cross-linked high amylose starches have been developed as excipients for the formulation of controlled-release solid dosage forms for the oral delivery of drugs. Advantages of this new class of excipients include cost-effectiveness, readily accessible industrial manufacturing technology, high active ingredient core loading and the possibility of achieving a quasi zero-order release for most drugs. In addition to the latter, other features distinguish cross-linked high amylose starches from other excipients used to prepare hydrophilic matrices. Among these are the absence of erosion, the limited swelling and the fact that increasing cross-linking degrees results in increased water uptake rate, drug release rate and equilibrium swelling. Thus the goal of the present study was to gain some insights into the mechanism of drug release control by matrices of cross-linked high amylose starch. Water transport kinetics and dimensional changes were studied in matrices placed in water at 37 degrees C by an image analysis technique. The results show that in the first 5 min, a gel layer is formed at the surface of the tablet, after which the gel front seems to halt its progression toward the center of the tablet. Water continues to diffuse through the front and to invade the core. As a consequence, this latter swells, with a predominance for radial swelling. Equilibrium swelling is reached over 3 days, when the water concentration in the tablet becomes homogeneous and the whole tablet gelifies. Solid-state 13C-NMR were acquired on cross-linked high amylose starch powders, tablets and hydrated tablets with varying cross-linking degrees. They show a predominance of the V-type single helix arrangement of amylose in the dry state irrespective of the cross-linking degree. Upon hydration, the homologues with a low cross-linking degrees show a transition from the V to the B-type double helix arrangement. It is therefore hypothesized that the capacity of amylose to undergo the V to B transition is an important factor in controlling water transport and drug release rate. Finally applications to different drugs are reviewed briefly. They illustrate the versatility of this technology as generic versions of zero order OROS drug (Efidac) and Fickian release conventional matrices (Voltaren SR) were developed and successfully tested in pilot clinical studies to be bioequivalent to the references. These studies further showed that cross-linked high amylose starch matrices have the lowest inter-subject variability among the systems tested and show a total absence of food effect.

Structure-properties relationship in cross-linked high-amylose starch for use in controlled drug release.

Cross-linked high-amylose starch (CLHAS), obtained by high-amylose starch cross-linking, was recently introduced as an excipient (Contramid) for monolithic dosage forms that are able to control drug release over 18-24 h. These control properties are related to tablet swelling and are strongly dependent on the degree of the cross-linking of CLHAS. The permeability of solutes through CLHAS hydrogels depends on the chemical structure of the polymer. The aim of this study was to obtain a better understanding of how modifications in CLHAS molecular structures at the level of long-range and short-range order during the cross-linking and processing conditions relate to the release properties of the CLHAS matrices. Structural parameters such as crystallinity contribute significantly to the physical and mechanical aspects of starch products. X-ray diffractometry, FTIR spectroscopy, dissolution tests in vitro, and mechanical hardness (of dry tablets) were found to be sensitive to the cross-linking degree (cld) variation. Best release properties and highest mechanical hardness were obtained from CLHAS matrices with low-to-moderate crystallinity, where the V- and the B-type structures coexist with amorphous regions. X-ray and FTIR profiles of dry CLHAS powders were found to be predictive for release properties of CLHAS tablets.

Development of biodegradable films from whey proteins by cross-linking and entrapment in cellulose.

When cross-linked by heating or by gamma-irradiation and entrapped in cellulose, whey proteins can generate insoluble biofilms with good mechanical properties and high resistance to attack by proteolytic enzymes. Interchain cross-linking of proteins generated an increase in the puncture strength, and a decrease in water vapor permeability. Gelatin was added in film formulation as a stabilizer to improve the puncture strength and film appearance. The structure of the biofilms was also analyzed. SDS-PAGE revealed that heating and gamma-irradiation produce an increase of the molecular weight of the cross-linked protein. Size exclusion chromatography showed a molecular mass of 40 kDa for un-cross-linked whey proteins, whereas for the soluble fractions of the cross-linked proteins, molecular distributions were between 600 and 3800 kDa for the heated proteins and between 1000 and 2000 kDa for gamma-irradiated proteins. No major alteration of the structural conformation of the proteins was observed by FTIR for biofilms obtained after heat treatment, whereas gamma-irradiation induced some modifications in the protein structure. X-ray diffraction analysis suggests that cross-linking by gamma-irradiation seems to modify the conformation of proteins, which became more ordered and more stable.

Mechanical and barrier properties of cross-linked soy and whey protein based films.

Sterilized biofilms based on soy protein isolate (SPI, S system) and a 1:1 mixture of SPI and whey protein isolate (WPI, SW system) were achieved through the formation of cross-links by means of gamma-irradiation combined with thermal treatments. The effect of the incorporation of carboxymethylcellulose (CMC) and poly(vinyl alcohol) was also examined. gamma-Irradiation combined with thermal treatment improved significantly the mechanical properties, namely, puncture strength and puncture deformation, for all types of films. Irradiated formulations that contain CMC behave more similarly as elastomers. CMC showed also significant improvements of the barrier properties, namely, water vapor permeability, for irradiated films of the S system and for non-irradiated films of the SW system.

Toxic effects of cadmium and copper on the isolated heart of dogfish shark, Squalus acanthias.

The adverse effects of heavy metal ions on the heart functions of lower vertebrates are largely unknown. In the present study, the effects of Cd2+, Cu2+, and Cu+ on the cardiac functions of the heart isolated from dogfish shark, Squalus acanthias, including the epicardial electrocardiogram, ventricular developed pressure (VDP), and heart beating rate, were studied. Cadmium (10 to 100 microM) significantly decreased VDP of the isolated shark hearts in a reversible manner. However, heart beating rate was not affected by cadmium. Cadmium also induced a transient modification of the amplitude and the form of the QRS complex. Cupric ion transiently increased VDP in a concentration-dependent manner, whereas cuprous ion (1 to 100 microM) did not markedly alter the cardiac functions of shark. Cupric or cuprous ions did not change heart beating rate and electrocardiogram at concentrations of 10 to 100 microM. Our results, for the first time, demonstrated the effects of cadmium on shark heart and indicated that the cardiac effects of copper are valence dependent. An elucidation of heavy metal effects on fish cardiac functions will help to understand the complex toxicological properties of heavy metals in different species and tissues, and will provide information for management of pollution control and marine resource protection.

Determination of serum myoglobin by the reverse passive hemagglutination assay (RPHA) and radioimmunoassay (RIA).

Serum myoglobin (Mb) is an important biological marker in the early diagnosis of the acute myocardial infarction (AMI). In a previous paper we showed that RPHA, which consists in the agglutination of anti-Mb antibody--coated erythrocytes by solutions containing Mb, is a simple, rapid and highly sensitive assay (approx. 1 ng/ml). 42 sera from AMI patients and 20 sera from healthy subjects were investigated by RPHA. The geometric mean (x/divided by SD) of serum Mb titers in AMI patients was 362 x/divided by 3.94, in the range of 64-8192 (the reciprocal dilutions) and in normal sera was 19 x/divided by 1.36, in the range of 16-32. The difference between the mean values was statistically significant at p < 0.001. Subsequently, serum Mb concentration was determined by RPHA and RIA in 51 AMI sera prelevated at various stages of the disease. A high degree of correlation was found between the two methods. Log Y (RPHA titers) = -0.9174 + 1.5879 log X (RIA); ESE: +/- 0.1947; r = 0.9601. In four AMI patients successive determinations were made at every 6 hours: serum Mb curves determined by RPHA and RIA had a parallel evolution and this was an additional argument for the validation of RPHA. Being quite simple and easy of execution RPHA is the most suitable method by which an early AMI diagnosis can be made.

NMR imaging of chitosan and carboxymethyl starch tablets: swelling and hydration of the polyelectrolyte complex.

The hydration and swelling properties of the tablets made of chitosan, carboxymethyl starch, and a polyelectrolyte complex of these two polysaccharides have been studied by NMR imaging. We studied the effect of pH and ionic strength on the swelling of the tablets and on the diffusion of fluid into the tablets in water and simulated physiological fluids. The pH value of the fluids exerts a more significant effect than their ionic strengths on the swelling of the tablets. The tablets are compared also with those made of cross-linked high amylose starch. The formation of complex helps to keep the integrity of the tablets in various media and render a slow and restricted swelling similar to that of the tablets of the cross-linked high amylase starch, which is significantly lower than the swelling of chitosan and of carboxymethyl starch. The capacities to modulate the release rate of drugs in different media are discussed by comparing the matrices and evaluating the preparation process of the complex. A sustained release of less soluble drugs such as aspirin in gastrointestinal fluids can be provided by the complex, due to the ionic interaction and hydrogen bonding between the drug and the biopolymer complex.

Fast adaptive uniformisation of the chemical master equation.

Within systems biology there is an increasing interest in the stochastic behaviour of biochemical reaction networks. An appropriate stochastic description is provided by the chemical master equation, which represents a continuous-time Markov chain (CTMC). The uniformisation technique is an efficient method to compute probability distributions of a CTMC if the number of states is manageable. However, the size of a CTMC that represents a biochemical reaction network is usually far beyond what is feasible. In this study, the authors present an on-the-fly variant of uniformisation, where they improve the original algorithm at the cost of a small approximation error. By means of several examples, the authors show that their approach is particularly well-suited for biochemical reaction networks.

Ceruloplasmin-induced aggregation of P19 neurons involves a serine protease activity and is accompanied by reelin cleavage.

The cytoarchitectural organization of the nervous system depends partly on extracellular serine proteases, including reelin. This 400K protein, which also exists as the N-terminally-derived 300K and 180K fragments, acts through binding to the lipoprotein receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR). Ceruloplasmin (CP), a multifunctional protein found in the circulation and also expressed on glial cells, was shown to bind to, and induce aggregation of neurons newly differentiated from P19 embryonic stem cells. This indicated a potential developmental role of CP in neuronal organization, possibly in relation with reelin and other extracellular serine proteases. Therefore, we analysed the effect of cell-impermeant, large spectrum, serine protease inhibitors on CP-induced neuroaggregation and studied reelin expression. Soybean trypsin inhibitor and aprotinin (SBTI+Apro) inhibited CP neuroaggregative action. Undifferentiated and neurally-differentiating cultures secreted the 400K reelin. The 180K fragment was present during and after differentiation whereas the 300K species was barely detectable. However, CP stimulated generation of the 300K in the differentiated neuronal cultures, and SBTI+Apro abolished this CP effect. Time course profiles and function-blocking antibody indicated that neuroaggregation does not depend on the generation of the 300K fragment or on reelin action. CP neuroaggregative action thus involves a pericellular serine protease, different from reelin. On the other hand, the CP stimulation of reelin cleavage is in line with a possible role of CP in nervous system development. Since P19 cells express ApoER2 and VLDLR, they can help understanding relationships existing between CP, reelin and intervening protease(s).

Importance of dynamic culture for evaluating osteoblast activity on dense silicon-substituted hydroxyapatite.

This paper reports an investigation on human osteoblast-like cells (SaOs-2) seeded onto pure hydroxyapatite (HA) and silicon-substituted HA (SiHA) tablets under static and dynamic culture conditions. The biological characterizations were conducted in classical static conditions in multi-wells plates, and in a perfusion bioreactor that permits continuous circulation of culture medium at 2 mL/h. The morphology, proliferation and differentiation of osteoblastic cells were examined for the two types of samples in the both culture conditions after 1, 3 and 8 days. Under dynamic conditions, cells cultured on SiHA surfaces showed a faster adhesion process and the formation of longer and thinner focal adhesions than in static conditions. The number of cells grown onto both ceramic surfaces was higher in dynamic conditions when compared with static conditions. Moreover, a higher activity of alkaline phosphatase was found for cells seeded under dynamic conditions. Our findings suggest that the application of perfusion culture system on cells cultured on dense substrates is valuable for predicting in vivo behaviour of cells on biomaterials.

Surface transformation of silicon-doped hydroxyapatite immersed in culture medium under dynamic and static conditions.

A comparative study of in vitro bioactivity of hydroxyapatite (HA) and silicon-doped hydroxyapatite (SiHA) has been carried out by immersion in a cell culture medium with or without fetal bovine serum during 14 days in static and dynamic conditions. A specific bioreactor was developed for the experiments in dynamic conditions. Ceramic surface transformations were characterized by electron microscopy, atomic force microscopy and X-ray photoelectron spectroscopy before and after immersion. The monitoring of calcium, phosphate and proteins in immersion medium was also done during the experiment. The two hydroxyapatite surfaces immersed in cell culture medium under dynamic conditions were found to be more probably covered by a new Mg-enriched Ca-deficient apatite layer than surfaces immersed under static conditions. These results suggest that dynamic procedure and medium with serum macromolecules seem to be more adequate to predict the in vivo activity of bioceramics. Moreover, SiHA presented a higher capacity of protein adsorption.