Measurement of Acetylcholine Release in Freely Moving Rats by Means of Automated Intracerebral Dialysis

The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC-assay system that comprises a cation exchanger, a post-column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 microM) of neostigmine resulted in gradually increasing amounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.     

Identification and Determination of 3,4-Dihydroxyphenylacetaldehyde, the Dopamine Metabolite, in In Vivo Dialysate from Rat Striatum

Abstract: 3,4-Dihydroxyphenylacetic acid (DOPAC) is commonly considered to be the main dopamine (DA) metabolite produced by monoamine oxidase (MAO); however, the initial product of DA oxidation is 3,4-dihydroxyphenylacetaldehyde (DOPALD). Owing to technical difficulties in detecting DOPALD from a biological matrix, no studies have so far been performed to measure brain levels of this aldehyde in vivo. In this work, using transstriatal microdialysis in freely moving rats, we identified DOPALD by HPLC coupled to a coulometric detector. In chromatograms obtained from microdialysis samples, DOPALD appeared as a peak with a retention time coincident with that of the standards obtained via enzymatic and chemical synthesis. On the other hand, DOPALD was undetectable ex vivo from rat striatal homogenates. This discrepancy is probably due to the preferential extraneuronal localization together with the high reactivity of the aldehyde, which is rapidly removed by the dialysis probe, whereas the ex vivo procedure allows its condensation and enzymatic conversion. Measurement of DOPALD levels as a routine procedure might represent a reliable tool to evaluate DA oxidative metabolism directly, in vivo. Moreover, parallel detection of DOPALD and DOPAC levels in brain dialysate may make it possible to distinguish between the activity of MAO and aldehyde dehydrogenase. DOPALD, like many endogenous aldehydes, has been shown to be toxic to the cell in which it is formed. Therefore, in vivo measurement of DOPALD levels could highlight new aspects in the molecular mechanisms underlying both acute neurological insults and neurodegenerative diseases.     

Cultured peritoneal mesothelial cells exhibit apical primary cilia

This study examined primary cilia on cultured human and rabbit peritoneal mesothelial cells (PMC) and investigated factors that influence ciliary expression. Primary cilia were examined with indirect immunocytochemistry, laser scanning confocal microscopy and scanning electron microscopy. Ciliary expression was evaluated in cultures with or without l-cysteine (0.25 mM) or exposure to Ca(2+)-free Krebs-Ringer solution supplemented with EGTA, 0.5 mM. This treatment disrupted cell monolayer integrity. Cilia were counted and normalized to total cell counts using NIH image. Primary cilia were identified on both human and rabbit PMC. Cells treated with l-cysteine expressed significantly more cilia compared with monolayers deprived of l-cysteine. Exposure to Ca(2+)-free Krebs-Ringer solution significantly reduced cilia (5.9+/-1.0%, n=7). Although ciliary expression could be augmented with l-cysteine, approximately 60% of human PMC and 84% of rabbit PMC did not exhibit cilia. Together, these data show that monolayers of PMC express apical cilia that can be augmented with l-cysteine, independently of increased cell density.     

Update on the challenging role of biofilms in peritoneal dialysis

Biofilms are commonly associated with an increased risk of patient infection. In peritoneal dialysis (PD), catheter associated infection, especially peritonitis, remains a clinically relevant problem. Although the presence of a biofilm is recognized in relapsing, repeat, and catheter-related peritonitis, it remains poorly characterized. In this review, an update on the role of biofilms in PD infections is presented. The emerging concept that host cells and tissue associated biofilms, in addition to the biofilms on the catheters themselves, contribute to the recalcitrance of infections is discussed. Furthermore, the evidence of biofilms on PD catheters, their developmental stages, and the possible influence of the PD environment are reviewed. The focus is given to ex vivo and in vitro studies that contribute to the elucidation of the interplay between host, microbial, and dialysis factors. The key issues that are still to be answered and the challenges to clinical practice are discussed.     

Molecular cut-off values of dialysis membranes for alginate and kappa-carrageenan oligosaccharides

The effective molecular weight cut-off values of dialysis membranes for carrageenan and alginate oligosaccharides were evaluated by gel permeation chromatography and nuclear magnetic resonance spectroscopy. For the different membranes tested, i.e. Medicell, Spectra Por 1000D and 3500D, the porous sizes are analogous to tri- and tetrasaccharides. A simple dialysis can be used to recover the majority of the oligosaccharides produced by a carrageenase or an alginate lyase digestion.     

N-Methylaspartate-Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release

The effect of N-methyl-D,L-aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration-dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20-100%. The effect of NMA appeared to be receptor-mediated, as coperfusion with D-2-amino-5-phosphonovaleric acid curtailed the NMA response by some 90%. The NMA-stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist D,L-cis-2,3-piperidine dicarboxylic acid. Although the NMA-evoked release of taurine and PEA was Ca2+-dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA-induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.     

Assembly of Plasmid DNA into Liposomes After Condensation by Cationic Lipid in Anionic Detergent Solution

A novel strategy to prepare negatively charged and small DNA-containing liposomes after condensation of plasmid DNA by a cationic lipid in deoxycholate micelle environment is described. The average diameter of resulting complexes was 62±8 nm. DNA-containing liposomes were then prepared by dialysis. The shape of the resulting liposomes was spherical. The average diameter and the surface charge of the liposomes were 86±6 nm and −24±3 mV, respectively. The plasmid DNA inside liposomes remained in a supercoiled form after incubation with DNase.     

Relationship Between Plasma and Brain Tryptophan in Pigs During Experimental Hepatic Coma Before and After Hernodialysis with Selective Membranes

Experimental acute liver ischemia in pigs induces an increment in plasma free tryptophan with decreased total tryptophan. Brain tryptophan is elevated in all brain areas. A slight, but significant increase of brain serotonin is demonstrated in the striatum only, while 5-HIAA (5-hydroxyindoleacetic acid) is significantly lower in the hypothalamus. Other brain areas do not show significant changes in serotonin and 5-HIAA levels. Neither the high plasma free tryptophan levels, nor the decreased sum of neutral competitive amino acids are consistent with such an elevation of brain tryptophan. Hemodialysis was carried out with two different kinds of membranes: cuprophan (with an efficient removal of molecules up to molecular weight 1300) and AN 69 polyacrylonitrile (efficient removal up to 15,000). Ammonia and aminoacid clearance are similar for both membranes. After AN 69, plasmatic free tryptophan and brain tryptophan are lower than after liver devascularization, but still higher than normal. Serotonin significantly increases in the cortex, midbrain and hypothalamus without concomitant rise of 5-HIAA levels. After cuprophan hemodialysis, plasma total tryptophan is lower than in normal and even comatose animals, whereas free tryptophan is normal. Intracerebral tryptophan is similar to AN 69 dialysed animals, but in the hypothalamus it is similar to nondialysed animals. Brain serotonin levels are not modified. 5-HIAA decreases in the hypothalamus. This finding suggests that middle molecules (which are not cleared out with cuprophan hemodialysis) are involved in the intracerebral transfer of tryptophan and the metabolism of serotonin, mainly in the hypothalamus.     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

The membrane dialysis bioreactor with integrated radial-flow fixed bed —a new approach for continuous cultivation of animal cells

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran^® carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l^?1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran^® carriers with 3–5 mm diameter showed an advantage compared to those with 1–2 mm diameter. For the 3–5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s^?1 and 0.75 mm s^?1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.