Nitric oxide decreases the sensitivity of pulmonary endothelial cells to LPS-induced apoptosis in a zinc-dependent fashion

We hypothesized that: (a) S-nitrosylation of metallothionein (MT) is a component of pulmonary endothelial cell nitric oxide (NO) signaling that is associated with an increase in labile zinc; and (b) NO mediated increases in labile zinc in turn reduce the sensitivity of pulmonary endothelium to LPS-induced apoptosis. We used microspectrofluorometric techniques to show that exposing mouse lung endothelial cells (MLEC) to the NO-donor, S-nitrosocysteine, resulted in a 45% increase in fluorescence of the Zn²⁺-specific fluorophore, Zinquin, that was rapidly reversed by exposure to the Zn²⁺ chelator, NNNN-tetrakis-(2-pyridylmethyl)ethylenediamine; TPEN). The absence of a NO-mediated increase in labile Zn²⁺ in MLEC from MT-I and -II knockout mice inferred a critical role for MT in the regulation of Zn²⁺ homeostasis by NO. Furthermore, we found that prior exposure of cultured endothelial cells from sheep pulmonary artery (SPAEC), to the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP) reduced their sensitivity to lipopolysaccharide (LPS) induced apoptosis. The anti-apoptotic effects of NO were significantly inhibited by Zn²⁺ chelation with low doses of TPEN (10 M). Collectively, these data suggest that S-nitrosylation of MT is associated with an increase in labile (TPEN chelatable) zinc and NO-mediated MT dependent zinc release is associated with reduced sensitivity to LPS-induced apoptosis in pulmonary endothelium.     

Nitrate uptake,nitrate reductase distribution and their relation to proton release in five nodulated grain legumes

Nitrate uptake, nitrate reductase activity (NRA) and net proton release were compared in five grain legumes grown at 0.2 and 2 mM nitrate in nutrient solution. Nitrate treatments, imposed on 22-d-old, fully nodulated plants, lasted for 21 d. Increasing nitrate supply did not significantly influence the growth of any of the species during the treatment, but yellow lupin (Lupinus luteus) had a higher growth rate than the other species examined. At 0.2 mM nitrate supply, nitrate uptake rates ranged from 0.6 to 1.5 mg N g(-1) d(-1) in the order: yellow lupin > field pea (Pisum sativum) > chickpea (Cicer arietinum) > narrow-leafed lupin (L angustifolius) > white lupin (L albus). At 2 mM nitrate supply, nitrate uptake ranged from 1.7 to 8.2 mg N g(-1) d(-1) in the order: field pea > chickpea > white lupin > yellow lupin > narrow-leafed lupin. Nitrate reductase activity increased with increased nitrate supply, with the majority of NRA being present in shoots. Field pea and chickpea had much higher shoot NRA than the three lupin species. When 0.2 mM nitrate was supplied, narrow-leafed lupinreleased the most H+ per unit root biomass per day, followed by yellow lupin, white lupin, field pea and chickpea. At 2 mM nitrate, narrow-leafed lupin and yellow lupin showed net proton release, whereas the other species, especially field pea, showed net OH- release. Irrespective of legume species and nitrate supply, proton release was negatively correlated with nitrate uptake and NRA in shoots, but not with NRA in roots.     

Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.     

Neuritogenesis: polarization of constitutive exocytosis by effectors of Rho-family GTPases?

The sprouting of neurites from a neuron represents a highly specialized form of cellular morphogenesis that must involve coordinated changes in two major cellular processes at two membrane locations: reorganization of the cytoskeleton and redirection of membrane traffic from the trans-Golgi network to the plasma membrane of the growth tip. How exactly are these two processes linked and how is spatial and temporal coordination achieved at the first instance of neurite sprouting? Recent advances may have already revealed some, if not most of the pieces in the puzzle. We discuss below, with some extrapolations, of what has recently come to light, and what more is needed to construct a coherent picture.     

Cytochrome P450 3A4-mediated oxidative conversion of a cyano to an amide group in the metabolism of pinacidil

The conversion of nitriles to amides is generally considered to be a hydrolytic process that does not involve redox chemistry. We demonstrate here that cytochrome P450 (CYP) is responsible for the conversion of the cyano group of pinacidil to the corresponding amide. The reaction in human liver microsomes was NADPH-dependent and was nearly completely inhibited by an anti-CYP3A4 antibody. Incubations of pinacidil with recombinant CYP enzymes confirm that CYP3A4 is the principal catalyst of this reaction. The kinetics of pinacidil amide formation by CYP3A4 yielded an apparent K(m) of 452 +/- 33 microM and k(cat) of 0.108 min(-1) (k(cat)/K(m) = 0.238 mM(-1).min(-1)). Incubation of pinacidil with CYP3A4 in the presence of (18)O(2) or H(2)(18)O showed that the amide carbonyl oxygen derived exclusively from molecular oxygen. The CYP3A4-mediated reaction also was supported by hydrogen peroxide when incubations were carried out in the absence of cytochrome P450 reductase and NADPH. The reaction can be explained by a nucleophilic attack of a deprotonated ferric peroxide intermediate (Fe(3+)-O-O(-)) on the carbon atom of the -C triple bond N triple bond to form an Enz-Fe(III)-O-O-C(=NH)R intermediate, followed by cleavage of the O-O bond to give pinacidil amide. This nucleophilic addition of an Fe(3+)-O-O(-) intermediate to a -C=N pi-bond in a P450 system resembles the analogous reaction catalyzed by the nitric oxide synthases.     

X-ray structures of Torpedo californica acetylcholinesterase complexed with (+)-huperzine A and (-)-huperzine B: structural evidence for an active site rearrangement

Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.     

Effects of volatile anesthetic on channel structure of gramicidin A

Volatile anesthetic agent, 1-chloro-1,2,2-trifluorocyclobutane (F3), was found to alter gramicidin A channel function by enhancing Na(+) transport (. Biophys. J. 77:739-746). Whether this functional change is associated with structural alternation is evaluated by circular dichroism and nuclear magnetic resonance spectroscopy. The circular dichroism and nuclear magnetic resonance results indicate that at low millimolar concentrations, 1-chloro-1,2,2-trifluorocyclobutane causes minimal changes in gramicidin A channel structure in sodium dodecyl sulfate micelles. All hydrogen bonds between channel backbones are well maintained in the presence of 1-chloro-1,2,2-trifluorocyclobutane, and the channel structure is stable. The finding supports the notion that low affinity drugs such as volatile anesthetics and alcohols can cause significant changes in protein function without necessarily producing associated changes in protein structure. To understand the molecular mechanism of general anesthesia, it is important to recognize that in addition to structural changes, other protein properties, including dynamic characteristics of channel motions, may also be of functional significance.     

Crystal structure of memapsin 2 (beta-secretase) in complex with an inhibitor OM00-3

The structure of the catalytic domain of human memapsin 2 bound to an inhibitor OM00-3 (Glu-Leu-Asp-LeuAla-Val-Glu-Phe, K(i) = 0.3 nM, the asterisk denotes the hydroxyethylene transition-state isostere) has been determined at 2.1 A resolution. Uniquely defined in the structure are the locations of S(3)' and S(4)' subsites, which were not identified in the previous structure of memapsin 2 in complex with the inhibitor OM99-2 (Glu-Val-Asn-LeuAla-Ala-Glu-Phe, K(i) = 1 nM). Different binding modes for the P(2) and P(4) side chains are also observed. These new structural elements are useful for the design of new inhibitors. The structural and kinetic data indicate that the replacement of the P(2)' alanine in OM99-2 with a valine in OM00-3 stabilizes the binding of P(3)' and P(4)'.     

A cyclin-dependent protein kinase homologue associated with the basal body domains in the ciliate Tetrahymena thermophila

The tight coupling between cell cycle progression and morphogenetic development in the unicellular ciliates presents a unique model system for examination of the roles of Cdks in developmental processes. We here describe the isolation and characterization of the first cyclin-dependent kinase (Cdk) homologue, TtCdk1, from Tetrahymena thermophila. TtCdk1 corresponds to the larger of the two polypeptides recognized by anti-PSTAIRE antibody in a whole cell lysate, which differ from each other in their affinity for yeast p13(suc1) protein. In contrast to the constant protein expression levels of typical eukaryotic Cdks, the TtCdk1 protein level fluctuates periodically over the vegetative cell cycle, reaching a maximum at the end of the cell cycle, correlating with its histone H1 kinase activity. Its association with the membrane-skeletal domains that surround mature, but not nascent, basal bodies in the cell cortex suggests that TtCdk1 plays a role in the regulation of cortical morphogenesis in T. thermophila. A partial TtCDK1 knockout cell line constructed through somatic biolistic transformation resulted in a reduction of the regularity of the rows of basal bodies plus an additional effect on chromatin condensation in both macro- and micronuclei. Unlike the situations in higher eukaryotic cells, no apparent effect on basal body duplication was found upon disruption of the TtCDK1 gene.