Driver behaviour during flashing green before amber: a comparative study.

The paper discusses the results of extensive measurements of drivers' stopping behaviour during signal programmes with and without flashing green before amber. Ten locations in Switzerland, Austria, and Germany were recorded with a video camera and analysed using an image-processing system. About 5000 cycles were documented. The analysis shows that the flashing green increases the number of early stops, as drivers tend to underestimate the duration of the time to the end of amber. Discrete choice models of the stopping behaviour are estimated for inclusion in suitable microsimulation models of traffic flow. The model results show that speed and distance to stop line, and their interaction (potential time to the stop line with unchanged speed) explain the stopping process.

Biodegradable recombinant human erythropoietin loaded microspheres prepared from linear and star-branched block copolymers: influence of encapsulation technique and polymer composition on particle characteristics.

Recombinant human erythropoietin (EPO) and fluorescein isothiocyanate labeled dextran (FITC-dextran) loaded microspheres were prepared by a modified W/O/W double-emulsion technique. Biodegradable linear ABA block copolymers consisting of poly(L-lactide-co-glycolide) A blocks attached to central poly(ethyleneoxide) (PEO) B blocks and star-branched AB block copolymers containing A blocks of poly(L-lactide) or poly(L-lactide-co-glycolide) and star-branched poly(ethyleneoxide) B blocks were investigated for their potential as sustained release drug delivery systems. Microsphere characteristics were strongly influenced by the polymer composition. In the case of the linear block copolymers, a reduced lactic acid content in a linear block copolymer yielded smaller particles, a lower encapsulation efficiency, and a higher initial drug release both in the case of EPO and FITC-dextran. The investigation of the effects of several manufacturing parameters on microsphere formation showed that the process temperature plays an important role. Microsphere formation in a +1 degrees C environment resulted in higher drug loadings without increasing the amount of residual dichloromethane inside the particles. Other parameters such as the homogenization of the primary W/O emulsion and of the W/O/W double-emulsion have less impact on microsphere characteristics. Branched block copolymers containing star-shaped PEO also showed potential for the preparation of drug loaded microspheres. A certain amount of glycolic acid in the copolymer was necessary for the successful preparation of non-aggregating microspheres at room temperature. Again, the processing temperature strongly affected particle characteristics. Microsphere preparation at +1 degrees C allows the formation of microspheres from a polymer not containing glycolic acid, a result which could not be achieved at room temperature. Moreover, compared to microsphere formation at room temperature, the effective FITC-dextran loading was increased. Concerning the EPO loaded microspheres, the amount of EPO aggregated was comparable to that using the linear ABA polymers. A continuous release of the protein from these star-shaped polymers could not be achieved. In conclusion, apart from microsphere preparation in a +1 degrees C environment the choice of the polymer represents the main factor for a successful entrapment of proteins into biodegradable microspheres.

Erythropoietin loaded microspheres prepared from biodegradable LPLG-PEO-LPLG triblock copolymers: protein stabilization and in-vitro release properties.

Biodegradable microspheres containing recombinant human Erythropoietin (EPO) were prepared from ABA triblock copolymers, consisting of hydrophobic poly(l-lactic-co-glycolic acid) A blocks and hydrophilic polyethylenoxide (PEO) B blocks. Different polymer compositions were studied for the microencapsulation of EPO using a modified double-emulsion process (W/O/W). The encapsulation efficiency for EPO, ranging from 72% to 99% was quite acceptable. The formation of high molecular weight EPO aggregates, however, was higher than in poly(d,l-lactide-co-glycolide) (PLG) microparticles. Using different excipients with known protein stabilizing properties, such as Bovine Serum Albumin (BSA), Poly-l-Histidine (PH), Poly-l-Arginine (PA) or a combination of PA with Dextran 40 (D40), the EPO aggregate content was significantly reduced to <5% of the encapsulated EPO. In contrast to PLG, ABA triblockcopolymers containing >7 mol % PEO, allowed a continuous release of EPO from microspheres for up to 2 weeks under in-vitro conditions. The release profile was comparable to FITC-Dextran 40 kDa (FD 40) loaded microspheres in the initial release phase, while EPO release was leveling off at later time points. BSA additionally prolonged the EPO release, while blends of PLG and PEO did not generate continuous EPO release profiles. LPLG-PEO-LPLG triblock-copolymers (35 mol % PEO; 30 kDa) in combination with 5% BSA yielded both an acceptable level of EPO aggregates and a continuous release profile under in-vitro conditions for up to 2 weeks. The formation of EPO aggregates at later time points is probably induced by acidic cleavage products of the biodegradable polymer and requires further optimization of the ABA polymer composition.

Recombinant human erythropoietin (rhEPO) loaded poly(lactide-co-glycolide) microspheres: influence of the encapsulation technique and polymer purity on microsphere characteristics.

Recombinant human erythropoietin (EPO) and fluorescein isothiocyanate-labelled dextran (FITC-dextran) loaded biodegradable microspheres were prepared from poly(lactide-co-glycolide) (PLG) by a modified spray-drying technique. This microencapsulation method was compared with the water-in-oil-in-water (w/o/w) double-emulsion method. As expected, microsphere morphology, particle size and particle size distribution strongly depended on the production process. The spray-drying method was found to have a number of advantages compared to the w/o/w double-emulsion technique. The content of residual dichloromethane (DCM) in the final product was significantly lower in case of the microspheres prepared by spray-drying. Concerning EPO loaded microspheres, spray-drying yielded higher encapsulation efficiencies. Although the microspheres obtained by spray-drying are subjected to intensive mechanical and thermal stress during the preparation, the amount of aggregates of EPO in PLG microspheres were not increased compared to the w/o/w technique. Depending on the manufacturing method, addition of cyclic DL-lactide dimers (referred to as monomers in the following) affected the in vitro release profiles of EPO and FITC-dextran from PLG microspheres. Using differential scanning calorimetry it was shown that these low molecular weight substances only seem to be present inside the microspheres produced by spray-drying. DL-Lactide significantly reduced the initial burst release of both EPO and FITC-dextran. While the following release period of EPO was not affected by the DL-lactide content, a more linear FITC-dextran release pattern could be achieved. It can be concluded that the spray-drying technique provides a number of advantages compared to the w/o/w method. The modulation of protein release using low molecular weight additives is of particular interest for parenteral depot systems.

Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry.

The site-specific glycan heterogeneity of human urinary erythropoietin was investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Owing to the small amount of protein available, a strategy combining optimal sensitivity and specificity was used. Erythropoietin was reduced, S-alkylated and digested with endoproteinase Lys C. The peptides were separated by reversed-phase high-performance liquid chromatography and the molecular masses of the peptides determined by MALDI-MS. The peptides were identified by comparing the experimental masses with the masses predicted from the cDNA derived amino acid sequence. Glycopeptides were identified from the mass spectra based on the peak pattern caused by the glycan heterogeneity. They were further characterized after treatment with neuraminidase and endoproteases. All N-glycosylation sites exhibited fucose-containing complex-type glycans. The N-glycosylation sites at Asn38 and Asn83 are mainly occupied by tetraantennary glycans, whereas Asn24 is occupied by a mixture of bi-, tri- and tetraantennary glycans. A molecular mass glycoprofile for each glycosylation site was established based on the relative peak intensities observed in the MALDI mass spectra of the desialylated glycopeptides.

Topogenesis of cytochrome oxidase subunit II. Mechanisms of protein export from the mitochondrial matrix.

Cytochrome c oxidase subunit II (COXII) in yeast mitochondria is synthesized as a precursor (preCOXII) and is sorted across the inner membrane, whereby both N and C termini become exposed to the intermembrane space. We describe here how this process can be experimentally dissected into a number of distinct stages. Our results demonstrate that the translation of COXII is not obligatorily coupled to translocation. Insertion into the inner membrane and export of the N- and C-terminal domains require an energized inner membrane. The export of COXII is independent of both maturation by the Imp1p protease and assembly into the cytochrome c oxidase complex. When linked to a mitochondrial matrix-targeting sequence, the N-terminal portion of preCOXII (fused to mouse dihydrofolate reductase) can be imported into the mitochondrial matrix. Following accumulation in the matrix, this chimeric protein can become exported across the inner membrane, delivering the N terminus into the intermembrane space where it undergoes processing by the Imp1p protease. This export process displays a number of similarities to bacterial protein export and supports the view that the principles of sorting are conserved from prokaryotes to eukaryotic organelles.

The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space.

The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted. Import and processing of precytochrome b2 (pb2), a precursor carrying a bipartite presequence, into the intermembrane space was also strongly dependent on matrix ATP. Preproteins, consisting of 220 or more residues of pb2 fused to dihydrofolate reductase, showed the same requirement for matrix ATP, whereas the import of shorter fusion proteins (up to 167 residues of pb2) was largely independent of matrix ATP. For those intermembrane-space-targeted proteins that did need matrix ATP, the dependence could be relieved either by unfolding these proteins prior to import or by introducing a deletion into the mature portion of the protein thereby impairing the tight folding of the cytochrome b2 domain. These results suggest the following: (a) The import of matrix-targeted preproteins, in addition to a membrane potential delta psi, requires matrix ATP [most likely to facilitate reversible binding of mitochondrial heat-shock protein 70 (mt-Hsp70) to incoming precursors], for two steps, securing the presequence on the matrix side of the inner membrane and for the completion of translocation; (b) in the case of intermembrane-space-targeted precursors with bipartite signals, the function of ATP/mt-Hsp70 is not obligatory, as components of the intermembrane-space-sorting pathway may substitute for ATP/mt-Hsp70 function (however, if a tightly folded domain is present in the precursor, ATP/mt-Hsp70 is indispensable); (c) unfolding on the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix-ATP/mt-Hsp70.

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space.

Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.

Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number.

A gene bank of a yeast wild type DNA in the high copy number vector YEp13 was screened for recombinant plasmids which suppress the mitochondrial RNA splice defect exerted by mutant M1301, a -1 bp deletion in the first intron of the mitochondrial COB gene (bI1). A total of 17 recombinant plasmids with similar suppressor activity were found. Restriction mapping and cross-hybridization of the inserts revealed that these 17 plasmids contain three different inserts, all lacking any extended sequence homology. Each of the inserts, when present in high copy number, has a similar suppressor activity: high in the presence of mutation M1301 in bI1, a group II intron, and low but significant with the presence of few mutants in bI2 and bI3 of the COB gene, both of which are group I introns.