Cigarette smoke-induced bronchoconstriction: causative agents and role of thromboxane receptors

Hong, Ju-Lun, and Lu-Yuan Lee. Cigarette smoke-inducedbronchoconstriction: causative agents and role of thromboxane receptors. J. Appl. Physiol. 81(5):2053-2059, 1996.Inhalation of cigarette smoke induces a biphasicbronchoconstriction in guinea pigs: the first phase is induced by acombination of cholinergic reflex and tachykinins, whereas the secondphase involves cyclooxygenase metabolites (J.-L. Hong, I. W. Rodger,and L.-Y. Lee. J. Appl. Physiol. 78:2260-2266, 1995). This study was carried out to further determinethe causative agents in the smoke and the types of prostanoid receptorsand endogenous prostanoids mediating the bronchoconstriction. Inhalation of 10 ml of high-nicotine cigarette smoke consistently elicited the biphasic bronchoconstriction in anesthetized and artificially ventilated guinea pigs. Pretreatment with hexamethonium (10 mg/kg iv) significantly reduced the first-phase bronchoconstriction but did not have any measurable effect on the second-phase response. Insharp contrast, gas-phase smoke did not elicit any bronchoconstrictive effect. Furthermore, when the animals were challenged with low-nicotine cigarette smoke, only a single second-phase response was evoked, accompanied by increases in thromboxane (Tx)B₂ (a stable metabolite ofTxA₂), prostaglandin (PG)D₂,PGF₂ in the bronchoalveolar lavage fluid. The bronchoconstrictive response induced by low-nicotine smoke was completely prevented by pretreatment with SQ-29548 (0.3 mg/kgiv), a TxA₂-receptor antagonist.These results indicate that 1)nicotine is the primary causative agent responsible for the first-phasebronchoconstriction and 2)nonnicotine smoke particulates evoke the release ofTxA₂,PGD₂, andPGF₂, which act onTxA₂ receptors on airway smoothmuscles and induce the second-phase response to cigarette smoke.

     

Penicillin acylase-catalyzed synthesis of β-lactam antibiotics in water-methanol mixtures: effect of cosolvent content and chemical nature of substrate on reaction rates and yields

The synthesis of four β-lactam antibiotics (penicillin G, pivaloyloxymethyl ester of penicillin G, ampicillin and pivampicillin) catalyzed byEscherichia coli penicillin acylase has been investigated in water-methanol mixtures. The enzyme reactions were either thermodynamically or kinetically controlled at the same conditions using phenylacetic acid andd--phenylglycine methyl ester as acyl donors and 6-aminopenicillanic acid and pivaloyloxymethyl 6-aminopenicillanic acid as acyl acceptors. It has been found that the influences of the cosolvent content on the reaction rates and synthetic yields are significantly different depending on the substrates used in the experiments. On the other hand, within certain ranges of the methanol content (up to ca. 40% (v/v) the residual activities of the enzymes in water-methanol mixtures were only slightly lower than those in aqueous media. To analyze the factors that determine the reaction rate in water-cosolvent mixtures, the effect of methanol on the apparent pK values of the substrates has been investigated, and a mathematical model has been developed on the basis of the assumption that the enzyme binds non-ionized substrates. Model simulation results indicate that the solvent effect on reaction rates is mainly attributed to the kinetic effects of changes in apparent pK values.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

A simple model of human foveal ganglion cell responses to hyperacuity stimuli

We developed a physiologically plausible model of the first steps of spatial visual information processing in the fovea of the human retina. With the predictions of this model we could support the hypothesis that, for moderate contrasts ( 40%), hyperacuity is mediated by the magnocellular (MC-) pathway. Despite the lower sampling density in the MC pathway, as compared to the parvocellular (PC-) pathway, the information that is transferred by the MC ganglion cells is sufficient to achieve thresholds comparable to those of human subjects in psychophysical tasks. This is a result of the much higher signal-to-noise ratio of the MC pathway cell signals. The PC pathway cells do not transfer enough information for hyperacuity thresholds.     

The polyamine-derived amino acid hypusine: its post-translational formation in eIF-5A and its role in cell proliferation

Summary The unusual amino acid hypusine [N -(4-amino-2-hydroxybutyl)lysine] is a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology, eIF-4D). It is formed posttranslationally and exclusively in this protein in two consecutive enzymatic reactions, (i) modification of a single lysine residue of the eIF-5A precursor protein by the transfer of the 4-aminobutyl moiety of the polyamine spermidine to its-amino group to form the intermediate, deoxyhypusine [N -(4-aminobutyl)lysine] and (ii) subsequent hydroxylation of this intermediate to form hypusine. The amino acid sequences surrounding the hypusine residue are strictly conserved in all eukaryotic species examined, suggesting the fundamental importance of this amino acid throughout evolution. Hypusine is required for the activity of eIF-5Ain vitro. There is strong evidence that hypusine and eIF-5A are vital for eukaryotic cell proliferation. Inactivation of both of the eIF-5A genes is lethal in yeast and the hypusine modification appears to be a requirement for yeast survival (Schnier et al., 1991 [Mol Cell Biol 11: 3105–3114]; Wöhl et al., 1993 [Mol Gen Genet 241: 305–311]). Furthermore, inhibitors of either of the hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxyhypusine hydroxylase, exert strong anti-proliferative effects in mammalian cells, including many human cancer cell lines. These inhibitors hold potential as a new class of anticancer agents, targeting one specific eukaryotic cellular reaction, hypusine biosynthesis.     

Flow cytometric analysis of antibody producing cells using double immunofluorescent staining

Summary Double fluorescent labeling, with fluorescein isothiocyanate (FITC)-labeled F(ab)₂ specific for the heavy chain and R-phycoerythrin (R-PE)-labeled F(ab)₂ specific for the light chain, was demonstrated as a convenient means for the accurate evaluation of a heterogeneous non-antibody-producing population. Furthermore, it could be used for monitoring the changes in each immunoglobulin (Ig) chain content of the cells during the batch culture, which will facilitate the study on antibody synthesis, assembly and secretion.     

Comparison of the structures of the cyclotheonamide A complexes of human alpha-thrombin and bovine beta-trypsin.

Thrombin, a trypsin-like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of alpha-thrombin (K(i) = 1.0 nM), but substantially more potent toward trypsin (K(i) = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of alpha-thrombin and beta-trypsin reported here focuses on structure-function relationships in general and the enhanced specificity of trypsin, in particular. The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 A resolution, respectively. The structures show that CtA occupies the active site with the Pro-Arg motif positioned in the S2 and S1 binding sites. The alpha-keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re-face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214-Gly 216 segment runs in a twisted antiparallel beta-strand manner with respect to the diaminopropionic acid (Dpr)-Arg segment of CtA. The Tyr 60A-Thr 60I insertion loop of thrombin makes a weak aromatic stacking interaction with the v-Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D-Phe of CtA, which also leads to different orientations of the side chains of D-Phe and the v-Tyr. The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174-Arg 175 by Gly 174-Gln 175 makes the S3 aryl site more polar because the Arg 175 side chain is directed away from thrombin and into the solvent, whereas Gln 175 is not. Because the site is occupied by the Dpr group of CtA, the occupancy of the S3 site is better in trypsin than in thrombin. In trypsin, the D-Phe side chain of CtA fits between Tyr 39 and Phe 41 in a favorable manner, whereas in thrombin, these residues are Glu 39 and Leu 41. The higher degree of specificity for trypsin is most likely the result of these substitutions and the absence of the fairly rigid Tyr 60A-Thr 60I insertion loop of thrombin, which narrows access to the active site and forces less favorable orientations for the D-Phe and v-Tyr residues.     

Expression of c-fos, c-jun and HSP70 mRNA in rat brain following high acceleration stress

Rats exposed to high +Gz forces in a small animal centrifuge (SAC) exhibit loss of neuronal function (isoelectric EEG), termed G-induced loss of consciousness (G-LOC). This phenomenon is presumably due to a reduction in cerebral blood flow (CBF) or ischemia. Ischemia induces various metabolic and physiologic changes including expression of immediate early genes (IEGs) in the brain. Expression of IEGs have been suggested to be reliable markers for neuronal response to external stimuli or stress. In the present study expression of IEGs c-fos, c-jun and stress response gene HSP70 were measured in the brains of rats subjected to six 30 s exposures of +22.5Gz in a small animal centrifuge. The level of c-fos, HSP70 and beta-actin mRNA were measured by both Northern blot and RT-PCR. Expression of c-jun was measured only by RT-PCR. Expression of c-fos and c-jun was significantly stimulated at 0.5, 15, 30 and 60 min post-centrifugation. The level of HSP70 mRNA was significantly higher only at 60 and 180 min post-centrifugation. Measurement of metabolities showed a significant increase in lactate and a decrease in Cr-P level at 30 s and 15 min post-centrifugation, respectively. Lactate, but not Cr-P and ATP levels were restored to control levels by 60 min post-centrifugation. It is concluded that the transient expression of c-fos, c-jun and HSP70 mRNA is stimulated by repeated ischemic/reperfusion episodes induced by high acceleration stress.