Soft microelectrode linear array for scanning electrochemical microscopy

A linear array of eight individual addressable microelectrodes has been developed in order to perform high-throughput scanning electrochemical microscopy (SECM) imaging of large sample areas in contact regime. Similar to previous reports, the soft microelectrode array was fabricated by ablating microchannels on a polyethylene terephthalate (PET) film and filling them with carbon ink. Improvements have been achieved by using a 5 μm thick Parylene coating that allows for smaller working distances, as the probe was mounted with the Parylene coating facing the sample surface. Additionally, the application of a SECM holder allows scanning in contact regime with a tilted probe, reducing the topographic effects and assuring the probe bending direction. The main advantage of the soft microelectrode array is the considerable decrease in the experimental time needed for imaging large sample areas. Additionally, soft microelectrode arrays are very stable and can be used several times, since the electrode surface can be regenerated by blade cutting. Cyclic voltammograms and approach curves were recorded in order to assess the electrochemical properties of the device. An SECM image of a gold on glass chip was obtained with high resolution and sensitivity, proving the feasibility of soft microelectrode arrays to detect localized surface activity. Finite element method (FEM) simulations were performed in order to establish the effect of diffusion layer overlapping between neighboring electrodes on the respective approach curves.     

On the specificity of tuna-directed primers in PCR-SSCP analysis of fish and meat

Single strand conformation polymorphism (SSCP) of an amplicon (148 bp) obtained by polymerase chain reaction (PCR) of the mitochondrial cytochrom b gene used to identify tuna species was studied with other fish and animal species. Single-stranded DNA (ssDNA) patterns of two to four strong bands were obtained with blue ling, carp, haddock, mackerel, mackerel shark, saithe, catfish, Alaska pollack, and skipjack which, however, differed from those obtained with tuna samples. Other fish species resulted in weak (cod, spined dogfish) or no ssDNA bands (Atlantic salmon, halibut, herring, pike-perch, plaice, redfish, sprat, trout). Samples from animals other than fish resulted in strong ssDNA bands differing from those of tuna and from each other (crayfish; cattle, European rabbit, fallow deer, hare, horse, red deer, roe deer; goose, turkey), in bands differing from tuna but not from each other (domestic goat/sheep, domestic pig/wild boar), or in weak bands (octopus, shrimp; chicken, duck). Increasing the stringency of PCR caused a more pronounced difference between strong and weak ssDNA bands. Inter-laboratory reproducibility of the method was good.     

Chemical activation and changes in surface morphology of poly(ε-caprolactone) modulate VEGF responsiveness of human endothelial cells

The high degree of clinical routine in percutaneous transluminal coronary angioplasty (PTCA) with and without stenting has not changed the fact that a large number of coronary heart disease patients are still affected by post-operative complications such as restenosis and thrombosis. Because re-endothelialization is the crucial aspect of wound healing after cardiovascular implant surgery, there is a need for modern biomaterials to aid endothelial cells in their adhesion and functional recovery post-stenting. This study systematically examines the potential of numerous chemical polymer modifications with regard to endothelialization. Poly(ε-caprolactone) (PCL) and its chemically activated forms are investigated in detail, as well as the impact of polymer surface morphology and precoating with matrix protein. Human umbilical vein endothelial cells (HUVECs) are used to characterize endothelial cell responses in terms of in vitro viability and adhesion. As a potential component in drug eluting implants, VEGF is applied as stimulus to boost endothelial cell proliferation on the polymer. In conclusion, plasma chemical activation of PCL combined with VEGF stimulation best enhances in vitro endothelialization. Examining the impact of morphological, chemical and biological modifications of PCL, this study makes an important new contribution towards the existing body of work on polymer endothelialization.     

Cocaine reward models: conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.     

Cellular localization and expression of the serotonin transporter in mouse brain

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using 35S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [125I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.     

Monitoring Cheddar cheese ripening by chemical indices of proteolysis 2. Peptide mapping of casein fragments by reverse-phase high-performance liquid chromatography

Summary The proteolysis of casein during cheese ripening was studied by reverse-phase (RP)-HPLC peptide mapping. Cheddar cheese from two different production plants were analysed during a 6-month ripening period at 4° C and 10° C. The elution profile obtained from cheese extracts soluble at pH 4.6 contained more than 120 peaks. These were grouped into four ranges of molecular mass (I<3000 Da; II>30 000 Da; III>10 000 Da; IV>3000 Da) by RP-HPLC of cheese extracts fractionated by ultrafiltration at different molecular mass cutoffs. The peptide patterns, especially in the molecular mass range below 3000 Da, were clearly dependent on ripening time and temperature, manufacturing history, and composition of the cheese. Several short chain peptides with less than ten amino acid residues were isolated, sequenced for identification, and assigned to the corresponding amino acid sequences of _s1-casein and-casein. The levels and ratios of these defined marker peptides seem to be well suited for in-depth characterisation of proteolysis and ripening of Cheddar cheese. This information is fundamental for studies on cheese origin, flavour, taste, and texture.

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Beurteilung des Reifungsverlaufes in Cheddar-Käse durch chemische Indikatoren des Eiweißabbaus 2. Peptid-Mapping von Caseinbruchstücken mit Umkehrphasen-Hochleistungschromatographie

Zusammenfassung Die Proteolyse des Caseins während der Käsereifung wurde durch Peptidtrennung mit RPHPLC studiert. Zwei verschiedene Cheddarkäse unterschiedlicher Herstellung wurden während einer sechsmonatigen Reifungszeit bei 4 °C und 10 °C analysiert. Das RP-HPLC-Elutionsprofil von pH 4,6-löslichen Käseextrakten wies mehr als 120 Komponenten auf. Diese wurden durch RP-HPLC von Käseextrakten, die mit verschiedenen Ausschlußgrenzen ultrafiltriert wurden, in vier Molekülmassen-Bereiche eingeteilt: I<3000; II>30 000; III>10 000; IV>3 000. Die Peptidmuster, insbesondere die im niedrigmolekularen Bereich (<3000), zeigten eindeutige Abhängigkeiten von Reifungszeit und Reifungstemperatur, Herstellungsprozeß und Zusammensetzung der Käse. Mehrere kurzkettige Peptide mit weniger als 10 Aminosäureresten wurden isoliert, zur Identifizierung sequenziert und den entsprechenden Aminosäuresequenzen von _s1-Casein und-Casein zugeordnet. Die Mengen und Mengenverhältnisse dieser definierten Leitpeptide scheinen für eine tiefergehende Charakterisierung des Eiweißabbaus und Reifungsverlaufes von Cheddarkäsen sehr gut geeignet zu sein. Für Studien über Herkunft, Geruch, Geschmack und Textur von Käse ist dies von entscheidender Bedeutung.