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Background and Purpose-Oxidant stress, especially in the premature, plays a major role in the pathogenesis of hypoxic-ischemic encephalopathies mostly manifested in the periventricular region. We studied the vasomotor mode of actions of the peroxidation product 15-F 2t -isoprostane (15-F 2t -IsoP) (8-iso-prostaglandin F 2␣ ) on periventricular region during development. Methods-Effects of 15-F 2t -IsoP on periventricular microvessels of fetal, newborn, and juvenile pigs were studied by video imaging and digital analysis techniques. Thromboxane formation and intracellular Ca 2ϩ were measured by radioimmunoassay and by using the fluorescent indicator fura 2-AM. Results-15-F 2t -IsoP-mediated constriction of periventricular microvessels decreased as a function of age such that in the fetus it was Ϸ2.5-fold greater than in juvenile pigs. 15-F 2t -IsoP evoked more thromboxane formation in the fetus than in the newborn, which was greater than that in the juvenile periventricular region; this was associated with immunoreactive thromboxane A 2 (TXA 2 ) synthase expression in the fetus that was greater than that in newborn pigs, which was greater than that in juvenile pigs. 15-F 2t -IsoP-induced vasoconstriction was markedly inhibited by TXA 2 synthase and receptor blockers (CGS12970 and L670596). Vasoconstrictor effects of the TXA 2 mimetic U46619 on fetal, neonatal, and juvenile periventricular microvessels did not differ. 15-F 2t -IsoP increased TXA 2 synthesis by activating Ca 2ϩ influx through non-voltage-gated channels in endothelial cells (SK&F96365 sensitive) and N-type voltage-gated channels (-conotoxin sensitive) in astrocytes; smooth muscle cells were not responsive to 15-F 2t -IsoP but generated Ca 2ϩ transients to U46619 via L-type voltage-sensitive channels. Conclusions-15-F 2t -IsoP causes periventricular brain region vasoconstriction in the fetus that is greater than that in the newborn, which in turn is greater than that in the juvenile due to greater TXA 2 formation generated through distinct stimulatory pathways, including from endothelial and astroglial cells. The resulting hemodynamic compromise may contribute to the increased vulnerability of the periventricular brain areas to oxidant stress-induced injury in immature subjects. (Stroke. 2000;31:516-525.)

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Microvascular degeneration is an important event in oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity. Because oxidant stress abundantly generates thromboxane A2 (TxA2), we tested whether TxA2 plays a role in retinal vasoobliteration of OIR and contributes to such vascular degeneration by direct endothelial cytotoxicity. Hyperoxia-induced retinal vasoobliteration in rat pups (80% O2 exposure from postnatal days 5-14) was associated with increased TxB2 generation and was significantly prevented by TxA2 synthase inhibitor CGS-12970 (10 mg x kg(-1) x day(-1)) or TxA2-receptor antagonist CGS-22652 (10 mg x kg(-1) x day(-1)). TxA2 mimetics U-46619 (EC50 50 nM) and I-BOP (EC50 5 nM) caused a time- and concentration-dependent cell death of neuroretinovascular endothelial cells from rats as well as newborn pigs but not of smooth muscle and astroglial cells; other prostanoids did not cause cell death. The peroxidation product 8-iso-PGF2, which is generated in OIR, stimulated TxA2 formation by endothelial cells and triggered cell death; these effects were markedly diminished by CGS-12970. TxA2-dependent neuroretinovascular endothelial cell death was mostly by necrosis and to a lesser extent by apoptosis. The data identify an important role for TxA2 in vasoobliteration of OIR and unveil a so far unknown function for TxA2 in directly triggering neuroretinal microvascular endothelial cell death. These effects of TxA2 might participate in other ischemic neurovascular injuries.

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A purine nucleoside phosphorylase from Aeromonas hydrophyla (AhPNP) was covalently immobilized in a pre‐packed stainless steel column containing aminopropylsilica particles via Schiff base chemistry upon glutaraldehyde activation. The resulting AhPNP‐IMER (Immobilized Enzyme Reactor, immobilization yield ≈50%) was coupled on‐line through a 6‐way switching valve to an HPLC apparatus containing an analytical or a semi‐preparative chromatographic column. The synthesis of five 6‐modified purine ribonucleosides was carried out by continuously pumping the reaction mixture through the AhPNP‐IMER until the highest conversion was reached, and then directing the reaction mixture to chromatographic separation. The conditions of the AhPNP‐catalyzed transglycosylations (2:1 ratio sugar donor:base acceptor; 10 mM phosphate buffer; pH 7.5; temperature 37 °C, flow rate 0.5 mL min−1) were optimized by a fractional factorial experimental design. Coupling the bioconversion step with the product purification in such an integrated platform resulted in a fast and efficient synthetic process (yield=52–89%; <10 mg) where sample handling was minimized. To date, AhPNP‐IMER has retained completely its activity upon 50 reactions in 10 months.magnified image

Microtubules are polymeric structures formed by the self-assembly of tubulin dimers. The growth and shrinkage of these dynamic arrays have a key role during the cell-proliferation process. This makes tubulin the molecular target of many anticancer drugs currently in use or under clinical trial. Their impressive success is limited by the onset of resistant tumour cells during the treatment, so new resistance-proof molecules need to be developed. Here we use molecular dynamics and free-energy calculations to study the network of interactions that allow microtubule formation. Modelling the protein-protein interface allows us to identify the amino acids responsible for tubulin-tubulin binding and thus to design peptides, which correspond to tubulin subsequences, that interfere with microtubule formation. We show that the application of molecular modelling techniques leads to the identification of peptides that exhibit antitubulin activity both in vitro and in cultured cells.