Intrapartum transfer of oxytocin across the human placenta: An ex vivo perfusion experiment.

INTRODUCTION: Investigation of the maternal to fetal transfer of oxytocin across the dually perfused term human placenta. METHODS: Human placentae obtained from term singleton pregnancies were utilized in a dual recirculating model of ex vivo placental perfusion. Six placentae from women delivering by elective cesarean at term were perfused, one blank and five with the test substance synthetic oxytocin (0.8 ng/mL) (OX) added to the maternal perfusate for 180 min. Antipyrine was used as positive control to validate overlap of the maternal and fetal circuits. The concentration of OX was determined by radioimmunoassay. RESULTS: A fall in maternal concentration of OX was seen throughout the experiment. At 90 min of perfusion a state of equilibrium was reached between maternal and fetal concentrations; however after 180 min the fetal concentration of OX was higher than that of the maternal. 31 % of the test substance was accounted for at the end of the experiment - suggesting OX protein binding and a high degree of oxytocinase activity. DISCUSSION: The ex vivo perfusion experiments revealed low transfer of OX to the fetal circuit below physiologically relevant concentrations.

Exploring Alzheimer molecular pathology in Down's syndrome cerebrospinal fluid.

BACKGROUND: Individuals with Down's syndrome (DS) develop early Alzheimer's disease (AD) with ß-amyloid (Aß) plaque pathology. The extra amyloid precursor protein (APP) gene copy in DS is believed to result in a 50% increase in Aß production, but it is unclear how this relates to the development of other AD hallmarks, including axonal degeneration and microglia cell activation, and to other neurological problems in DS, including disturbed sleep regulation. OBJECTIVE: To evaluate if cerebrospinal fluid (CSF) biomarkers for cerebral amyloidosis, axonal degeneration, microglial activation and sleep regulation were altered in young and old patients with DS, and if these biomarkers were related to altered Aß and APP metabolism, reflected by CSF levels of different Aß and APP peptides. METHODS: CSF from DS patients (n=12) and healthy controls (n=20) were analyzed for Aß peptides (Aß1-42, AßX-38/40/42), secreted APP species (sAPPα/ß), biomarkers for AD-like axonal degeneration [total tau (T-tau), phosphorylated tau], microglial activation (YKL-40, CC chemokine ligand 2) and orexin-A, which is a peptide involved in sleep regulation. We compared biomarker levels between groups and tested for relations between biomarkers, disease stage and age. RESULTS: Several of the markers were specifically increased in DS, including AßX-40, sAPPα and sAPPß. Οrexin-A was significantly decreased in DS and correlated with Aß and sAPP. Orexin-A decreased with age in DS, while T-tau and YKL-40 increased with age. CONCLUSION: Down's patients have increased APP and Aß production and increased microglial activation with age. The orexin-A metabolism is disturbed in DS and may be linked to APP and Aß production. Biomarker studies of DS may contribute to our understanding of the amyloidogenic and neurodegenerative process in AD.

Unimpaired postprandial pancreatic polypeptide secretion in Parkinson's disease and REM sleep behavior disorder.

BACKGROUND: Pancreatic polypeptide is released immediately after food ingestion. The release is operated by vagal-abdominal projections and has therefore been suggested as a test for vagal nerve integrity. Pathoanatomical and clinical studies indicate vagal dysfunction in early Parkinson's disease (PD). METHODS: We assessed the postprandial secretion of pancreatic polypeptide and motilin in healthy controls (n = 18) and patients with idiopathic rapid-eye-movement sleep behavior disorder (iRBD, n = 10), a potential premotor stage of PD, as well as in drug-naive (n = 19) and treated (n = 19) PD patients. RESULTS: The postprandial pancreatic polypeptide secretion showed a physiological pattern in all groups and even an enhanced response in drug-naive PD and iRBD. Motilin concentrations correlated with pancreatic polypeptide concentrations. CONCLUSIONS: Postprandial pancreatic polypeptide secretion is not a suitable test for vagal nerve integrity in PD. The unimpaired pancreatic polypeptide response in iRBD and PD might be explained by partially intact vagal-abdominal projections or compensatory mechanisms substituting a defective neuronal brain-gut axis.

Proteomics/peptidomics tools to find CSF biomarkers for neurodegenerative diseases.

Neurodegenerative diseases are characterized by premature neuronal loss in specific brain regions. During the past decades our knowledge on molecular mechanisms underlying neurodegeneration has increased immensely and resulted in promising drug candidates that might slow down or even stop the neuronal loss. These advances have put a strong focus on the development of diagnostic tools for early or pre-clinical detection of the disorders. In this review we discuss our experience in the field of neuroproteomics/peptidomics, with special focus on biomarker discovery studies that have been performed on CSF samples from well-defined patient and control populations.

Proteomic analysis using protein chips to detect biomarkers in cervical and amniotic fluid in women with intra-amniotic inflammation.

Intra-amniotic inflammation (IAI) may cause preterm birth with poor neonatal out-come. To identify novel biomarkers for IAI, we analyzed amniotic and cervical fluid samples from 27 patients with signs of threatening preterm birth with or without IAI by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Seventeen proteins were significantly overexpressed in amniotic fluid from IAI cases and more often in women with preterm labor than those with rupture of membranes. Five of these were identified as human neutrophil protein 1-3, calgranulin A and B.

Analysis of proteins from a glioma cell line by using micro-scale solution isoelectric focusing in combination with liquid chromatography/tandem mass spectrometry.

In this study we have investigated whether micro-solution isoelectric focusing (microsol-IEF) can be used as a pre-fractionation step prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) and if extensive sample purification of the different fractions is required. We found that, in spite of the high concentrations of buffer and detergents, no clean up of the digested microsol-IEF fractions was necessary before analysis by LC/MS/MS. We also concluded that it is possible to identify at least twice as many proteins in a glioma cell lysate with the combination of microsol-IEF and LC/MS/MS than with LC/MS/MS alone. Furthermore, most of the proteins that were identified from one microsol-IEF fraction by using analytical narrow-range two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were also identified by LC/MS/MS. Finally, we used the combination of microsol-IEF and LC/MS/MS to compare two sample preparation methods for glioma cells and found that several nuclear, mitochondria, and endoplasmic reticulum proteins were only present in the sample that had been subjected to lipid extraction by incubating the homogenized cells in chloroform/methanol/water.

Proteomics and peptidomics in neuroscience. Experience of capabilities and limitations in a neurochemical laboratory.

The increasing use of proteomics has created a basis for new strategies to develop methodologies for rapid identification of protein patterns in living organisms. It has also become evident that proteomics has other potential applications than protein and peptide identification, e.g. protein characterization, with the aim of revealing their structure, function(s) and interactions of proteins. In comparative proteomics studies, the protein expression of a certain biological system is compared with another system or the same system under perturbed conditions. Global identification of proteins in neuroscience is extremely complex, owing to the limited availability of biological material and very low concentrations of the molecules. Moreover, in addition to proteins, there are number of peptides that must also be considered in global studies on the central nervous system. In this overview, we focus on and discuss problems related to the different sources of biological material and sample handling, which are part of all preparatory and analytical steps. Straightforward protocols are desirable to avoid excessive purification steps, since loss of material at each step is inevitable. We would like to merge the two worlds of proteomics/peptidomics and neuroscience, and finally we consider different practical and technical aspects, illustrated with examples from our laboratory.

Proteome analysis of conditioned medium from cultured adult hippocampal progenitors.

It is known that proliferation and survival of neural stem/progenitor cells in vitro not only depend on exogenous factors, but also on autocrine factors secreted into the conditioned medium. It is also well known that the identification of bioactive proteins secreted into the conditioned medium poses a substantial challenge. Recently, neural stem/progenitor cells were shown to secrete a survival factor, cystatin C, into the conditioned medium. Here, we demonstrate an approach to identify other low molecular weight proteins in conditioned medium from cultured adult rat hippocampal progenitor cells. A combination of preparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry was utilized in the analysis. We were able to identify a number of proteins, which include Rho-guanine nucleotide dissociation inhibitor 1, phosphatidylethanolamine binding protein (PEBP), also termed Raf-1 kinase interacting protein, polyubiquitin, immunophilin FK506 binding protein 12 (FKBP12) and cystatin C. The presence of PEBP and FKBP12 in conditioned medium was confirmed immunologically. All nestin-positive progenitor cells showed immunoreactivity for antibodies against PEBP and FKBP12. To our knowledge we are the first to use this preparative proteomic approach to search for stem cell factors in conditioned medium. The method could be used to identify novel bioactive proteins secreted by stem/progenitor cells in vitro. Identification of bioactive proteins in vitro is of potential importance for the understanding of the regulatory mechanisms of the cells in vivo.