Peri-marketing surveillance of lamotrigine in The Netherlands: doctors' and patients' viewpoints.

Purpose: Evaluation of daily clinical practice in prescribing lamotrigine in refractory epilepsy patients.

Methods: A collaborative, retrospective, peri-marketing study was performed in in- and out-patients attending one of the three Dutch epilepsy centres. Analysis of both patients' and doctors' information was performed in 520 patients using questionnaires and medical files.

Synthesis of the Streptomyces lividans maltodextrin ABC transporter depends on the presence of the regulator MalR.

During growth with maltotriose or amylose, Streptomyces lividans and Streptomyces coelicolor A3(2) synthesize a maltodextrin uptake system with highest specificity for maltotriose. The transport activity is absent in mutants of S. coelicolor A3(2) lacking a functional MalE binding protein.

Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry.

Loss of phosphoric acid is the most effective fragmentation reaction of pSer- and pThr-containing phosphopeptides of small size (up to 10-15 residues) in low-energy collision-induced dissociation. Therefore, tandem mass spectrometry with neutral loss scanning was evaluated for its utility to analyze protein phosphorylation using protein kinase A (PKA) catalytic subunit, which is phosphorylated at Thr197 and Ser338, as an example. Analysis of tryptic digests of phosphoproteins by tandem mass spectrometry with scanning for neutral loss of phosphoric acid resulted in spectra with poor signal-to-noise ratio, mainly because of the large size of the phosphopeptides formed (>2 kDa).

Five-membered ring formation in unimolecular reactions of peptides: a key structural element controlling low-energy collision-induced dissociation of peptides.

Unimolecular fragmentation reactions of peptides in low-energy collision-induced dissociation are reviewed in the mechanistic context of five-membered ring formation. This structure of intermediates or of fragment ions is recognized as a key element that governs unimolecular peptide fragmentation within the structural framework determined by the peptide backbone and its side-chains. A collection of collision-induced dissociation reactions is presented covering (i) b-ion formation, (ii) the fragmentation of N-terminally acylated peptides, (iii) neutral loss of the C-terminal amino acid in alkali or silver cationized peptides, (iv) the fragmentation of isoAsp-containing peptides and (v) the fragmentation of negatively charged Asp- or Glu-containing peptides.

Analysis of isoaspartate in peptides by electrospray tandem mass spectrometry.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic L-Asp/L-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of L-Asp by L-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (L-Asp/L-isoAsp)-X bond and of the X-(L-Asp/L-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased.

Binding characteristics of CebR, the regulator of the ceb operon required for cellobiose/cellotriose uptake in Streptomyces reticuli.

The Streptomyces reticuli Avicelase (cellulase, Cell) hydrolyzes crystalline cellulose to cellooligomers, cellobiose and cellotriose which are taken up by mycelia via an ABC transport system (Ceb) induced during growth with cellobiose or cellulose. The cebR gene located upstream of the cebEFG operon was cloned in Escherichia coli in frame with six histidine-encoding codons. The resulting purified fusion protein was shown to bind to a motif of 23 bp, including a perfect 18-bp palindrome situated upstream of the cebEFG.

MsiK-dependent trehalose uptake in Streptomyces reticuli.

During cultivation in the presence of trehalose Streptomyces reticuli expresses an inducible, highly specific trehalose uptake system that is absent in Streptomyces lividans. A palmitated trehalose-binding protein was identified in the cytoplasmic membrane of mycelia, extracted with the detergent Triton X-100 and purified using a trehalose affinity matrix. Immunological studies showed that within S.

Microfibril-associated protein 4 is present in lung washings and binds to the collagen region of lung surfactant protein D.

We have purified a glycoprotein from bovine lung washings using affinity chromatography on a maltose-affinity column. On SDS-polyacrylamide gel electrophoresis the protein showed a molecular mass of 36 kDa in the reduced state and 66 kDa in the unreduced state. On gel permeation chromatography the apparent molecular mass was 250 kDa.

Characterization of the binding protein-dependent cellobiose and cellotriose transport system of the cellulose degrader Streptomyces reticuli.

Streptomyces reticuli has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH2-terminal part of an intracellular beta-glucosidase (BglC).

The ABC maltose transporter.

Bacterial ATP-binding cassette (ABC) transporters and their homologues in eukaryotic cells form one of the largest superfamilies known today. They function as primary pumps that couple substrate translocation across the cytoplasmic membrane to ATP hydrolysis. Although ABC transporters have been studied for more than three decades, the structure of these multi-component systems is unknown, and the mechanism of transport is not understood.

Amitogenic alginates: key to first clinical application of microencapsulation technology.

Microencapsulation refers to a technique of immunoisolation by coating single cells or tissue with a semipermeable membrane. By combining microencapsulation with a specific tissue culturing method, iso-, allo-, and xenotransplantation of parathyroid tissue has been achieved without immunosuppression in a long-term animal model. Prior to its clinical use, continued analyses of the alginate, used as a coating substance, determined its mitogenic properties.

First successful xenotransplantation of microencapsulated human parathyroid tissue in experimental hypoparathyroidism: long-term function without immunosuppression.

Owing to the complexity of the parathyroid hormone's metabolic interactions, clinical hypoparathyroidism is one of the most difficult of all endocrine disorders to treat. Therefore, causative treatment of this disorder by transplantation of parathyroid glands is highly desirable. We have recently documented the long-term in vivo function of iso- and allotransplanted rat parathyroid tissue without systemic immunosuppression in an animal model.

The Streptomyces ATP-binding component MsiK assists in cellobiose and maltose transport.

Streptomyces reticuli harbors an msiK gene which encodes a protein with an amino acid identify of 90% to a corresponding protein previously identified in Streptomyces lividans. Immunological studies revealed that S. lividans and S.

Isotransplantation of microencapsulated parathyroid tissue in rats.

Permanent hypoparathyroidism is one of the most difficult of all endocrine disorders to treat medically. While autotransplantation of parathyroid tissue is clinically established, allotransplantation without immunosuppression is still at the level of animal experiments. Although persons affected by hypoparathyroidism are facing a clearly reduced quality of life, hypoparathyroidism rarely is a life threatening condition.

Transplantation of parathyroid tissue in experimental hypoparathyroidism: in vitro and in vivo function of parathyroid tissue microencapsulated with a novel amitogenic alginate.

Microencapsulation of tissues is an alternative to postoperative immunosuppression in transplantation. In 1994 iso-, allo- and xenotransplantation of microencapsulated parathyroid tissue was achieved in vivo. However, continued analysis of the coating substance (an alginate) determined mitogenic properties.

A lipid-anchored binding protein is a component of an ATP-dependent cellobiose/cellotriose-transport system from the cellulose degrader Streptomyces reticuli.

During cultivation in the presence of cellobiose or crystalline cellulose, Streptomyces reticuli expresses an inducible uptake system that transports cellobiose (K(m), 4 microM), cellotriose and, to a lesser degree, cellotetraose and cellopentaose. Cellobiose uptake is dependent on ATP and inhibited by N-ethylmaleimide. A binding protein was identified in its palmitylated form in the cytoplasmic membrane of mycelia.

Biochemical identification of a lipoprotein with maltose-binding activity in the thermoacidophilic Gram-positive bacterium Alicyclobacillus acidocaldarius.

Growth of the thermoacidophilic Gram-positive bacterium Alicyclobacillus acidocaldarius strain ATCC 27009 on maltose resulted in the increased production of a protein with apparent molecular mass of 40 kDa. By metabolic labelling with 14C-palmitic acid, the 40-kDa protein was identified as a lipoprotein. The protein exhibited maltose-binding activity at pH 3.

TrkH and its homolog, TrkG, determine the specificity and kinetics of cation transport by the Trk system of Escherichia coli.

The corrected sequence of the trkH gene of Escherichia coli predicts that the TrkH protein is a hydrophobic membrane protein of 483 amino acid residues, of which 41% are identical to those of the homologous and functionally analogous TrkG protein. These two proteins form the transmembrane component of the Trk system for the uptake of K+. Each protein alone is sufficient for high-level Trk activity.

Reduced N-acetylaspartate content in the frontal part of the brain in patients with probable Alzheimer's disease.

The fully relaxed water signal was used as an internal standard in a STEAM experiment to calculate the concentrations of the metabolites: N-acetylaspartate (NAA), creatine + phosphocreatine [Cr + PCr], and choline-containing metabolites (Cho) in the frontal part of the brain in 12 patients with probable Alzheimer's disease. Eight age-matched healthy volunteers served as controls. Furthermore, T1 and T2 relaxation times of the metabolites and signal ratios: NAA/Cho, NAA/[Cr + PCr], and [Cr + PCr]/Cho at four different echo times (TE) and two different repetition times (TR) were calculated.

NAD+ binding to the Escherichia coli K(+)-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport.

The nucleotide sequence of trkA, a gene encoding a surface component of the constitutive K(+)-uptake systems TrkG and TrkH from Escherichia coli, was determined. The structure of the TrkA protein deduced from the nucleotide sequence accords with the view that TrkA is peripherally bound to the inner side of the cytoplasmic membrane. Analysis by a dot matrix revealed that TrkA is composed of similar halves.

Altered expression of P-glycoprotein and cellular adhesion molecules on human multi-drug-resistant tumor cells does not affect their susceptibility to NK- and LAK-mediated cytotoxicity.

Drug resistance has been associated with resistance to NK- and LAK-cell-mediated cytotoxicity. We evaluated this issue in human cell lines, using multiple myeloma cells (8226) and 2 multi-drug-resistant (MDR) sublines selected using doxorubicin (8226/Dox40) and mitoxantrone (8226/MR40). In parallel, we studied the human breast carcinoma cell line series MCF7, MCF7/D40 and MCF7/Mitox.