Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology.

Chitosan gels with well-controlled morphology were prepared by an enzymatic chitosan gelation process using in-situ production of ammonia through the urea enzymatic hydrolysis by urease. The reaction kinetics were studied by four complementary methods (rheological, colorimetric, flowing and pH-metric measurements) to define an advanced gelation pH (and the corresponding advanced gelation time) enabling to obtain homogeneous chitosan gels with well-controlled morphology and strong enough to be suitable for a future membrane preparation. A comparative study of chitosan gelation kinetics in water and aqueous chitosan solution highlighted differences mainly related to ammonia consumption when chitosan was present. The key parameters evidenced for the control of the gelation kinetics were the temperature and the urease concentration. The operating parameters required to elaborate gels with sufficient mechanical resistance to be handled from 2.5% w/v chitosan concentration were defined: a 3.5umL-1 urease concentration, a 75mM urea concentration and a temperature of 5°C.

An innovative membrane bioreactor for methane biohydroxylation.

In this study, a membrane bioreactor (MBR) was developed for efficient, safe microbial methane hydroxylation with Methylosinus trichosporium OB3b. This innovative MBR, which couples a bioreactor with two gas/liquid macroporous membrane contactors supplying the two gaseous substrates (methane and oxygen) was operated in fed-batch mode. The feasibility and the reproducibility of this new biohydroxylation process were first demonstrated. The mass transfer within this MBR was twice that observed in a batch reactor in similar conditions. The productivity reached with this MBR was 75±25mgmethanol(gdrycell)(-1)h(-1). Compared to the literature, this value is 35times higher than that obtained with the only other fed-batch membrane bioreactor reported, which was run with dense membranes, and is comparable to those obtained with bioreactors fed by bubble-spargers. However, in the latter case, an explosive gas mixture can be formed, a problem that is avoided with the MBR.

A new silver based composite material for SPA water disinfection.

A new composite material based on alumina (Al2O3) modified by two surface nanocoatings - titanium dioxide (TiO2) and silver (Ag) - was studied for spa water disinfection. Regarding the most common microorganisms in bathing waters, two non-pathogenic bacteria Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram positive) were selected as surrogates for bacterial contamination. The bactericidal properties of the Al2O3-TiO2-Ag material were demonstrated under various operating conditions encountered in spa water (temperature: 22-37 °C, presence of salt: CaCO3 or CaCl2, high oxygen content, etc.). Total removal of 10(8) CFU mL(-1) of bacteria was obtained in less than 10 min with 16 g L(-1) of material. Best results were observed for both conditions: a temperature of 37 °C and under aerobic condition; this latest favouring Reactive Oxygen Species (ROS) generation. The CaCO3 salt had no impact on the bactericidal activity of the composite material and CaCl2 considerably stabilized the silver desorption from the material surface thanks to the formation of AgCl precipitate. Preliminary tests of the Al2O3-TiO2-Ag bactericidal behaviour in a continuous water flow confirmed that 2 g L(-1) of material eliminated more than 90% of a 2.0 × 10(8) CFU mL(-1) bacterial mixture after one water treatment recycle and reached the disinfection standard recommended by EPA (coliform removal = 6 log) within 22 h.

A new biosynthetic tracer for the inline measurement of virus retention in membrane processes: part I--Synthesis protocol.

In this study, a new biosynthetic tracer was developed to characterize the virus retention dynamics of membrane systems. This new tracer is a modified bacteriophage obtained by the grafting of enzymatic probes to an MS2 bacteriophage, one of the smallest non-pathogenic bacteria viruses, with an average diameter of about 30 nm. A protocol for the synthesis and purification of this new tracer was developed in this work. The production of this biosynthetic tracer was first qualitatively shown by a chromatographic characterization and an enzymatic test. The average number of probes grafted per phage was then quantified for three batches of tracers made from the same native phage suspension and the same batch of enzymatic probes. This quantification demonstrated the reproducibility of the synthesis protocol developed.

A new biosynthetic tracer for the inline measurement of virus retention in membrane processes: part II. Biochemical and physicochemical characterizations of the new tracer.

In a previous work, a reproducible procedure to produce a new biosynthetic tracer was developed. This new tracer is an MS2 bacteriophage with enzymatic probes grafted on its surface, which can induce enzymatic activity of the tracer. In this paper, the biochemical and physicochemical characteristics of this new tracer are determined. A protocol was developed to determine the specific enzymatic activity kcat(TRACER) of the tracer, which was found to be 2.93±0.78×10(4) min(-1) on average. Physicochemical characterizations of this new tracer showed that it is representative of viruses and may thus be used as a virus surrogate to assess the virus retention of membrane systems inline. Notably, the mean diameter and molecular weight of the tracer were found to be respectively 64.1±0.3 nm and 12,140±3654 kDa, which are within the size and molecular weight ranges of pathogenic viruses carried by water. The tracer surface was also studied and revealed the considerable porosity of the grafted probe layer, with a mean porosity of 88%, which could explain why the zeta potential of the tracers (-14.34±1.66 mV) was nearly the same as that of the native MS2 phages. Finally, a comparison between filtration of the reference microorganism used for membrane performance assessment (the MS2 phage) and the tracer suspensions showed the same filtration behaviour.

Size-controlled synthesis and characterization of thiol-stabilized gold nanoparticles.

Size-controlled synthesis of nanoparticles of less than a few nanometers in size is a challenge due to the spatial resolution limit of most scattering and imaging techniques used for their structural characterization. We present the self-consistent analysis of the extended x-ray absorption fine-structure (EXAFS) spectroscopy data of ligand-stabilized metal nanoclusters. Our method employs the coordination number truncation and the surface-tension models in order to measure the average diameter and analyze the structure of the nanoparticles. EXAFS analysis was performed on the two series of dodecanethiol-stabilized gold nanoparticles prepared by one-phase and two-phase syntheses where the only control parameter was the gold/thiol ratio xi, varied between 6:1 and 1:6. The two-phase synthesis resulted in the smaller particles whose size decreased monotonically and stabilized at 16 A when xi was lowered below 1:1. This behavior is consistent with the theoretically predicted thermodynamic limit obtained previously in the framework of the spherical drop model of Au nanoparticles.

ERG30, a VAP-33-related protein, functions in protein transport mediated by COPI vesicles.

Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat protein-endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)-which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP. The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH(2) terminus contains a predicted coiled-coil motif. We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods. Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles. It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.

Neu differentiation factor stimulates phosphorylation and activation of the Sp1 transcription factor.

Neu differentiation factors (NDFs), or neuregulins, are epidermal growth factor-like growth factors which bind to two tyrosine kinase receptors, ErbB-3 and ErbB-4. The transcription of several genes is regulated by neuregulins, including genes encoding specific subunits of the acetylcholine receptor at the neuromuscular junction. Here, we have examined the promoter of the acetylcholine receptor epsilon subunit and delineated a minimal CA-rich sequence which mediates transcriptional activation by NDF (NDF-response element [NRE]). Using gel mobility shift analysis with an NRE oligonucleotide, we detected two complexes that are induced by treatment with neuregulin and other growth factors and identified Sp1, a constitutively expressed zinc finger phosphoprotein, as a component of one of these complexes. Phosphatase treatment, two-dimensional gel electrophoresis, and an in-gel kinase assay indicated that Sp1 is phosphorylated by a 60-kDa kinase in response to NDF-induced signals. Moreover, Sp1 seems to act downstream of all members of the ErbB family and thus may funnel the signaling of the ErbB network into the nucleus.

Isolation and characterization of the highly phosphorylated repeat domain of distinct heavy neurofilament subunit (NF-H) isoforms.

1. Recent examination of the hypothesis that distinctly phosphorylated NF-H isoforms exist in different types of neurons revealed that the extent of phosphorylation of the heavy neurofilament polypeptide of bovine ventral root motor neurons is markedly higher than that of dorsal root neurons. 2. In the present study we employed endoproteinase ASP-N for isolating the Lys-Ser-Pro (KSP)-rich domain of NF-H, which contains most of the NF-H phosphorylation sites. 3. Treatment of NF-H with ASP-N endoproteinase results in a cascade of products, the last of which is a polypeptide with apparent molecular weight of 120 kDa. Amino terminal sequence and amino acid composition analysis revealed that this fragment contains the KSP-rich domain of NF-H. 4. Treatment of ventral and dorsal root NF-H with ASP-N endoproteinase and analysis of the phosphoserine contents of the resulting 120 kDa fragments revealed that the 120 kDa fragment of ventral root NF-H is significantly more phosphorylated than that of dorsal root NF-H. 5. These findings show that the difference in extent of phosphorylation of ventral and dorsal root NF-H is due at least partly to the KSP-rich domain of NF-H.

Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.

The ErbB family includes two receptors, ErbB-1 and ErbB-3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB-2. Unlike ErbB-1 and ErbB-2, the intrinsic tyrosine kinase of ErbB-3 is catalytically impaired. By using interleukin-3-dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB-3 is devoid of any biological activity but both ErbB-1 and ErbB-2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB-3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter-receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB-3-containing complexes, especially the ErbB-2/ErbB-3 heterodimer, are more active than ErbB-1 complexes. Nevertheless, ErbB-1 signaling displays dominance over ErbB-3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen-activated protein kinases ERK and c-Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase-defective ErbB-3.

Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H.

1. Previous immunohistochemical studies led to the suggestion that distinctly phosphorylated neurofilament isoforms exist in different types of neurons. We have recently examined this hypothesis by direct biochemical experiments, which revealed that the heavy neurofilament protein NF-H of bovine ventral root cholinergic neurons is more acidic and markedly more phosphorylated than that of bovine dorsal root neurons. 2. In the present study we employed this system to study the degree to which distinctly phosphorylated NF-H isoforms differ in the extents to which they can be phosphorylated and dephosphorylated in vitro. This was performed utilizing alkaline phosphatase and protein kinase PK40ERK, which is specific to serines of Lys-Ser-Pro (KSP) repeats. The results obtained reveal that: 3. The more extensively phosphorylated ventral root NF-H is dephosphorylated more rapidly than dorsal root NF-H. 4. Ventral root NF-H and dorsal root NF-H in their native form are both poor substrates of PK40ERK. 5. Following dephosphorylation, ventral root and dorsal root NF-H are phosphorylated extensively and differentially by this kinase. Under these conditions, PK40ERK catalyzes the incorporation of, respectively, 4.2 +/- 1.3 and 2.8 +/- 0.6 mol of phosphate per molecule of ventral root NF-H and dorsal root NF-H. The ratio of phosphates incorporated into ventral root NF-H to those incorporated into dorsal root NF-H is 1.46 +/- 0.17. 6. These findings support the hypothesis that different classes of neurons contain distinctly phosphorylated neurofilaments and show that ventral root and dorsal root neurons are a useful model system for studying the distinct characteristics of neurofilament phosphorylation in different types of neurons.

Antibodies to different isoforms of the heavy neurofilament protein (NF-H) in normal aging and Alzheimer's disease.

Sera of normal controls and of patients with neurological diseases contain antineurofilament antibodies. Recent studies suggest that biochemically and immunologically distinct subclasses of neurofilaments occur in different types of neurons. Alzheimer's disease (AD), the major cause of dementia, is associated with a marked degeneration of brain cholinergic neurons. In the present work we characterized the repertoire and age dependence of antineurofilament antibodies in normal sera and examined whether the degeneration of cholinergic neurons in AD is associated with serum antibodies directed specifically against the neurofilaments of mammalian cholinergic neurons. This was performed by immunoblot assays utilizing neurofilaments from the purely cholinergic bovine ventral root neurons and from the chemically heterogeneous bovine dorsal root neurons. Antibodies to the heavy neurofilament protein NF-H were detected in normal control sera. Their levels were significantly higher in older (aged 70-79) than in younger (aged 40-59) subjects. These antibodies bound similarly to bovine ventral root and dorsal root NF-H and their NF-H specificity was unchanged during aging. In contrast, the levels of IgG in AD sera that are directed against ventral root cholinergic NF-H were higher than those directed against the chemically heterogeneous dorsal root NF-H. Immunoblot experiments utilizing dephosphorylated ventral root and dorsal root NF-H and chymotryptic fragments of these molecules revealed that AD sera contain a repertoire of antimamalian NF-H IgG. A subpopulation of these antibodies binds to phosphorylated epitopes that are specifically enriched in ventral root cholinergic NF-H and that are located on the carboxy terminal domain of this molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinctly phosphorylated neurofilaments in different classes of neurons.

Recent immunohistochemical experiments revealed that specific anti-neurofilament monoclonal antibodies yield distinct patterns in different types of neurons. This led to the suggestion that neurofilaments are a family of heterogeneous molecules whose occurrence and distribution are a function of cell type. In the present study we examined the hypothesis that this heterogeneity is due to differences in the extent of phosphorylation of neurofilament proteins in distinct types of neurons. In view of the large number of potential phosphorylation sites on the heavy neurofilament protein (NF-H), we focused on this protein and examined its extent of phosphorylation in different types of neurons. This was performed using neurofilaments isolated from axons of the cholinergic bovine ventral root motor neurons and of the chemically heterogeneous bovine dorsal root neurons. Two-dimensional gel electrophoresis revealed that the isoelectric point of ventral root NF-H (pl 5.10) was approximately 0.2 pl units more acidic than that of dorsal root NH-F. This difference was abolished by treating the neurofilaments with alkaline phosphatase, suggesting that the excess negative charge of ventral root NF-H is due to increased levels of phosphorylation. Amino acid analysis confirmed that the phosphoserine content of ventral root NF-H (27.2 +/- 2.5% of the serines) is markedly higher than that of dorsal root NF-H (15.5 +/- 6.2% of the serines). These findings provide a novel system for studying the biochemistry and function of distinctly phosphorylated neurofilaments in different types of neurones.

A rapid and sensitive heterogeneous immunoelectrochemical assay using disposable electrodes.

In this novel enzyme-tagged immuoelectrochemical assay, disposable carbon felt discs serve both as electrodes and as the heterogeneous solid phase. Antibodies are immobilized on the carbon felt via a diaminoalkane-biotin-avidin-biotin bridge. Alkaline phosphatase is used as a label. Bound antibodies are monitored by following the electro-oxidation of aminophenol, produced enzymatically from p-amino-phenyl phosphate by the immobilized alkaline phosphatase at the electrode surface. A model system designed for determination of mouse IgG concentration yielded a calibration curve ranging from 10 pg/ml to 100 micrograms/ml. This assay can be performed rapidly and a single determination completed within 20 minutes. The system is useful also for rapid quantitation of a small number (approximately 80 organisms per ml) of bacteria.