Domperidone upregulates dopamine receptor expression and stimulates locomotor activity in larval zebrafish (Danio rerio).

Dopamine (DA) plays a significant role in cognition, motor function and social behavior. The objectives of this study were to (1) quantify the temporal expression of transcripts (DA receptors, transporters and tyrosine hydroxylase) associated with DA signaling during early stages of zebrafish development and (2) determine their expression profiles following treatment with a D2 receptor antagonist domperidone (DMP). We also assessed locomotor behavior following treatment with DMP using alternating periods of light and dark (ie, dark photokinesis), as DA plays a key role in behavior. Relative expression levels of transcripts that were investigated and related to the DA system were detected after the first 24 hours postfertilization (hpf). Some DA receptor transcripts (eg, drd4c) increased in abundance earlier in the embryo compared with other receptors (eg, drd3), suggesting that DA receptor paralogs may have unique roles in development. Treatment of larvae with DMP resulted in the upregulation of DA receptor transcripts (ie, drd1, drd7, drd4b, drd4c) and DA transporter 1 (ie, slc6a3), and it is hypothesized that upregulation of genes related to the DA system is a compensatory neurophysiological response to DA receptor antagonism. Larval activity during dark photokinesis (measured by distance traveled) was also elevated by DMP. We hypothesize that behavioral responses observed with DMP may be related to the regulation of deep brain photoreception in zebrafish (Danio rerio) (ZF) larvae by DA.

Affinity binding of a vampire bat plasminogen activator to SEC resins.

DSPAalpha1 is a recombinant form of the vampire bat plasminogen activator which we have produced in mammalian cell culture. During the development of a recovery process for DSPAalpha1 we observed an unexpected binding interaction between this protein and several types of gel filtration chromatography resins. Under typical operating conditions using neutral pH buffers, we found that DSPA flows through the sizing resin and is fractionated, as expected, according to its molecular size. However, DSPA applied under certain acidic conditions (

Application of new analytical technology to the production of a "well-characterized biological".

The role of new analytical technology in the development of the concept of a "Well Characterized Biological" is to provide suitable methodology that allows the characterization of even the most complex protein sample so that a consistent manufacturing process can be established. Glycoproteins are among the most challenging of products to characterize because of extreme sample microheterogeneity due to the carbohydrate moieties. As an example of the appropriate use of new analytical technology this review will examine the steps necessary to demonstrate that a glycoprotein is a "Well Characterized Biological". The key to characterization of complex protein samples lies in the use of appropriate combinations of the different methods that analyse the sample from substantially orthogonal and independent directions. An important advantage of capillary electrophoresis (HPCE) in this application is the complementarily of the technique with reversed phase HPLC (RPLC). Thus mixtures of variants of a polypeptide that are difficult to separate by RPLC can often be readily resolved by HPCE. Both separation techniques are well suited to the analysis of peptide maps, although RPLC is particularly powerful when used in combination with on-line electrospray mass spectrometry (ESI-MS) which allows for the effective ionization and detection of even high MW glycopeptides. In this sense the ESI-MS is an ideal detector for on-line mass detection after a RPLC separation of medium MW fragments (300 to 6000 emu) that are typically present in an enzyme digest of a protein. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOFMS) is a valuable technique for off-line characterization of CE fractions due to the high sensitivity of the method and its tolerance of samples with moderate levels of salt. The development of an effective protocol for the analysis of glycoform populations of intact glycoproteins by a combination of HPCE and off-line MALDI-TOFMS wil be demonstrated by the successful analysis of two highly heterogeneous glycoproteins, ovalbumin and Desmodus Salivary Plasminogen Activator (DSPA).

Application of multidimensional affinity high-performance liquid chromatography and electrospray ionization liquid chromatography-mass spectrometry to the characterization of glycosylation in single-chain plasminogen activator. Initial results.

Preliminary results are presented using a combination of affinity chromatography, reversed-phase HPLC and electrospray ionization mass spectrometry to produced peptide maps for N-linked, O-linked and non-glycosylated peptides from an endoproteinase LysC digest of DSPA alpha 1, a recombinant DNA derived glycoprotein. Although the system was used to identify a number of major N-linked structures, notably complex biantennary structures attached to asparagine 362, no O-linked glycopeptides from the possible 4 attachment sites were identified. The system did, however, demonstrate the feasibility of the approach and the applicability of the instrumental system.

Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator.

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.

Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization--time of flight mass spectrometry to the characterization of single-chain plasminogen activator.

The analysis of recombinant Desmodus salivary plasminogen activator (DSPA alpha 1), a heterogeneous glycoprotein, is demonstrated through the use of high-performance liquid chromatography (HPLC), high-performance capillary electrophoresis (HPCE), liquid chromatography-electrospray mass spectrometry (LC--ES-MS), and matrix-assisted laser desorption ionization--time of flight mass spectrometry (MALDI--TOF-MS). The proteins is analyzed at three specific levels of detail: the intact protein, proteolytic digests of the protein, and fractions from the proteolytic digest. A method for "on-column" collection of HPLC fractions for subsequent transfer and analysis by HPCE and MALDI--TOF-MS is shown.