MICRODIALYSIS AND LUMINESCENT PROBE - ANALYTICAL AND CLINICAL ASPECTS

Immobilized enzymes are widely used in the clinical laboratory to assay several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable, disposable and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow sensors based on the use of nylon tube coil or epoxy methacrylate column as solid support. For in-vivo determination a suitable microdialysis probe inserted directly into brain or blood allows continuous measurement of extracellular lactate levels by means of a bioluminescent flow detector system. This procedure performs more measurements in the same time interval than other systems (HPLC), e.g. to give a detailed description of the effects of ischemia, or other pathological events, on lactate concentration in the brain

Nitrile Oxides in Medicinal Chemistry-4. Chemoenzymatic Synthesis of Chiral Heterocyclic Derivatives

The two enantiomers of 3-bromo-5-(hydroxymethyl)-DELTA-2-isoxazoline (1) and 2-phenyl-5-(hydroxymethyl)-isoxazolidin-3-one (9) have been prepared in enantiomeric excess higher than 90\% by hydrolysis of the corresponding butyrates under the catalysis of lipase PS, which was the most selective catalyst of the enzymes tested. The pairs of enantiomers of 1 and 9 were transformed into the chiral forms of the potent muscarinic ligands 3 and 5. The results obtained with the homogeneous set of esters 6, 7, 10a-d evidence a strong dependence of reaction rate and enantioselectivity of the lipase PS-catalyzed transformations upon both the size of the acyl moiety and the shape of the group carrying the alcoholic part of the ester. In the series of esters 10a-d, the best results were obtained with butyrate 10b. Quite interestingly, on passing from the butyrate of 1 to that of 9, the value of the enantiomeric ratio remained remarkably high but the enantiopreference switched from R to S. In between lies the butyrate of 2-methyl-5-(hydroxymethyl)isoxazolidin-3-one {[(+/-)-6] which was barely recognized by lipase PS and yielded alcohol (R)-(-)-2 in a modest enantiomeric excess.

Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility

none12Background & Aims: The role of autoimmunity underlying paraneoplastic gut dysmotility remains unsettled. Because anti-Hu antibodies may impair enteric neuronal function, we tested whether anti-HuD-positive sera from patients with paraneoplastic gut dysmotility or commercial anti-HuD antibodies activated the apoptotic cascade in a neuroblastoma cell line and cultured myenteric neurons. Methods: Anti-HuD antibodies from patients with severe paraneoplastic gut dysmotility were characterized by immunofluorescence and immunoblot. SH-Sy5Y neuroblasts and cultured myenteric neurons were exposed to sera containing anti-HuD antibodies or 2 commercial anti-HuD antibodies. Cells were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique to evaluate apoptosis. Immunofluorescence was used to identify activated caspase-3 and apaf-1, along with microtubule-associated protein 2. Results: In SH-Sy5Y cells, the percentage of TUNEL-positive nuclei observed after exposure to anti-HuD-positive sera (32% ± 7%) or anti-HuD antibodies (23% ± 2%) was significantly greater than that of control sera or fetal calf serum (P < 0.001). The time-course analysis showed a significantly greater number of apoptotic neuroblastoma cells evoked by the 2 commercial anti-HuD antibodies at 24, 48, and 72 hours versus controls. The number of TUNEL-positive myenteric neurons exposed to anti-HuD antibodies (60% ± 14%) was significantly greater than that of fetal calf serum (7% ± 2%; P < 0.001). Apaf-1 and caspase-3 immunolabeling showed intense cytoplasmic staining in a significantly greater proportion of cells exposed to anti-HuD-positive sera or to commercial anti-HuD antibodies compared with controls. Conclusions: Anti-HuD antibodies evoked neuronal apoptosis that may contribute to enteric nervous system impairment underlying paraneoplastic gut dysmotility. Apaf-1 activation suggests participation of a mitochondria-dependent apoptotic pathway.noneDE GIORGIO R; BOVARA M; BARBARA G; CANOSSA M; SARNELLI G; DE PONTI F; STANGHELLINI V; TONINI M; CAPPELLO S; PAGNOTTA E; NOBILEORAZIO E; CORINALDESI R.DE GIORGIO, Roberto; Bovara, M; Barbara, G; Canossa, M; Sarnelli, G; DE PONTI, F; Stanghellini, V; Tonini, M; Cappello, S; Pagnotta, E; Nobileorazio, E; Corinaldesi, R

FXIII levels and genotypes in myocardial infarction: a potential novel prognostic biomarker?

Low FXIII levels impairs wound healing, causes cardiac rupture and remodeling after myocardial infarction (MI). Molecular imaging demonstrates FXIII in the MI area. We recently recognized acute FXIII drops in the first post-MI days, and this was genotype dependent. Our objective is to investigate if FXIII drop, and/or late recovery, could be a novel MI marker and if a FXIII prognostic threshold could exist. We recruited 250 MI patients from Coronary Care Unit of Hospital-University of Ferrara: 72% male; 65.5±12.5yy; STEMI 78.5%; PCI 95%. We performed 1-year follow-up and the combined end-point was re-MI, death, heart failure, stroke, u-angina (MACE). FXIII level was assessed by a commercial kit [Instrumentation Laboratory, Italy] at the recruitment (t0) and every 24-hour for 5 days (t1-5) post-MI. Control samples were drawn at 30-day (t30) to have steady state levels. Patients were V34L-genotyped. Globally, t0 had 100.5±33.5 FXIII mean level; the lowest value was at t4-5 (85.3±27.3) and t30 was 99.4±25.7. Stratifying data by genotype, L-carriers had the highest fall when compared to VV-genotype. Stratifying patients by FXIII-(t4-5) median level (73.2%) there was a non-significant MACE overrepresentation among low FXIII levels (31% vs 15%). Conversely, by using ROC-FXIII-cutoff (81.3%), the difference was significant (30% vs 9.5%; P=0.03). We conclude that, despite excellent therapy for MI, post-MI MACE still remain critical influencing survival. Understanding whether FXIII level monitoring may be useful to select cases with poor clinical outcome could pave the way to utilize FXIII as tailored treatment to improve myocardial healing, recovery of functions and survival