Glutathione-mediated mineralization of 14C-labeled 2-amino-4,6-dinitrotoluene by manganese-dependent peroxidase H5 from the white-rot fungus Phanerochaete chrysosporium

Manganese-dependent peroxidase (MnP) H5 from the white-rot fungus Phanerochaete chrysosporium, in the presence of either Mn(II) (10 mM) or GSH (10 mM), was able to mineralize ¹⁴C-U-ring-labeled 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) up to 29% in 12 days. When both Mn(II) and GSH were present, the mineralization extent reached 82%. On the other hand, no significant mineralization was observed in the absence of both Mn(II) and GSH, suggesting the requirement of a mediator [either Mn(II) or GSH] for the degradation of 2-A-4,6-DNT by MnP. Using electron spin resonance (ESR) techniques, it was found that the glutathionyl free radical (GS^•) was produced through the oxidation of GSH by MnP in the presence as well as in the absence of Mn(II). GS^• was also generated through the direct oxidation of GSH by Mn(III). Our results strongly suggest the involvement of GS^• in the GSH-mediated mineralization of 2-A-4,6-DNT by MnP. Received: 18 February 2000 / Received revision: 24 May 2000 / Accepted: 26 May 2000     

Iridoids of garrya elliptica as plant growth inhibitors

Extracts from catkins of Garrya elliptica inhibit the growth of wheat embryos. The components responsible for this activity have been identified as the iridoids geniposide and geniposidic acid together with their aglucones.     

CD80/CD86 costimulation regulates acute vascular rejection

Xenotransplantation may provide the only solution to the shortage of human donor organs. Although hyperacute rejection associated with xenotransplantation can now be overcome, acute vascular rejection (AVR) remains a primary barrier to xenotransplantation. To date, standard immunosuppressive agents fail to block AVR or prolong xenograft survival. The present study was undertaken to determine the role of CD80/CD86 costimulatory molecules in regulating AVR. Lewis rat hearts were transplanted heterotopically into wild-type or IL-12, CD80- or CD86-deficient C57BL/6 mice. Wild-type recipients were treated with CD80 or CD86 neutralizing Ab with and without daily cyclosporin A (CsA, 15 mg/kg). Transplanted hearts in untreated wild-type recipients were rejected on postoperative days (POD) 17-21 and showed cell-mediated rejection (CMR) and AVR pathologies. In contrast, transplanted hearts in IL-12 and CD80 recipients or wild-type recipients treated with CD80 neutralizing Ab were rapidly rejected on POD 5 and 6 with AVR pathology. Interestingly, hearts transplanted into CD86 knockout recipients or wild-type recipients treated with CD86 neutralizing Ab underwent CMR on POD 17. Finally, blockade of CD86 but not CD80 rendered xenograft recipients sensitive to daily CsA therapy, leading to indefinite xenograft survival. To conclude, we demonstrate that AVR can be overcome by blocking the CD86 costimulatory pathway. Furthermore, we demonstrate that CD80 and CD86 have opposing roles in regulation of xenotransplantation rejection, where CD80 drives CMR and attenuates AVR while CD86 drives AVR. Most strikingly, indefinite xenograft survival can be achieved by suppressing AVR with CD86 neutralization in combination of CsA therapy, which inhibits CMR.     

Lithium enhances secretion from large dense-core vesicles in nerve growth factor-differentiated PC12 cells

Considerable attention has been focused on the therapeutic role of lithium (Li) in bipolar disorders. Although no consensus has emerged, Li presumably influences the behavior of neurons that regulate mood and behavior. Using PC12 cells to study cellular and molecular actions of Li, we previously reported that Li modulates the expression of proteins associated with large dense-core vesicles (LDCVs; organelles typically containing monoamines, neuropeptides and other cargo proteins). The current investigation indicates that this enhanced expression of LDCV proteins correlates with an altered secretory phenotype in Li-treated cells. Immunoblotting detects significant increases in the cellular content and secretion of the LDCV cargo proteins chromogranin B and secretogranin II. Amperometry reveals an increase of spike number elicited by K+-depolarization of Li-treated cells but no change of spike amplitude or kinetics. Electron microscopy reveals no significant change in LDCV number per unit area in Li-treated cells. However, there is a significant increase (about 15%) in the diameter of LDCVs after Li. Thus, Li induces changes in the properties of LDCVs that culminate in augmented regulated secretion in nerve growth factor-differentiated PC12 cells. These results extend our understanding of Li-dependent changes of cellular function that may be germane to the therapeutic action of Li.     

Chemotaxis and random motility in unsteady chemoattractant fields: a computational study

We discuss a generic computational model which captures the effects of transient chemoattractant concentration on the chemotactic motility of individual cells. The model solves the appropriate unsteady chemoattractant transport equation using finite differences, while simultaneously executing biased random walks representing individual cells. The simulations were implemented for a 2D homogeneous domain, and two case studies were considered. In the first case study, we consider a single-point source at the origin of the domain which produces chemoattractant, while other cells execute biased random walks toward this point source. We observe that for continuous chemoattractant production, chemoattractant diffusivity has no effect on cell motility, as measured by the mean of time to reach the source. However, in the case of pulsed random production with a specific average duty cycle, the mean time-to-contact is generally minimal with respect to chemoattractant diffusivity over a moderate range of diffusivities. In the second case study, two mobile cells which simultaneously secrete chemoattractant are initially placed a certain distance apart and are then allowed to execute biased random walks. Our model shows that a pulsed random protocol for chemoattractant production facilitates the two cells "finding" one another compared to continuous production. From this case study we also learn that there exists a range of moderate chemoattractant diffusivities for which the mean time-to-contact is minimal when cells both produce/detect chemoattractant and chemotactically migrate. Using these case studies, we discuss how transience in chemoattractant concentration becomes important in characterizing the effectiveness of chemotaxis.     

Cooperative binding of midazolam with testosterone and alpha-naphthoflavone within the CYP3A4 active site: a NMR T1 paramagnetic relaxation study

Recent studies have indicated that CYP3A4 exhibits non-Michaelis-Menten kinetics for numerous substrates. Both homo- and heterotropic activation have been reported, and kinetic models have suggested multiple substrates within the active site. We provide some of the first physicochemical data supporting the hypothesis of allosteric substrate binding within the CYP3A4 active site. Midazolam (MDZ) is metabolized by CYP3A4 to two hydroxylated metabolites, 1'- and 4-hydroxymidazolam. Incubations using purified CYP3A4 and MDZ showed that both alpha-naphthoflavone (alpha-NF) and testosterone affect the ratio of formation rates of 1'- and 4-hydroxymidazolam. Similar to previous reports, alpha-NF was found to promote formation of 1'-hydroxymidazolam, while testosterone stimulated formation of 4-hydroxymidazolam. NMR was used to measure the closest approach of individual MDZ protons to the paramagnetic heme iron of CYP3A4 using paramagnetic T(1) relaxation measurements. Solutions of 0.2 microM CYP3A4 with 500 microM MDZ resulted in calculated distances between 7.4 and 8.3 A for all monitored MDZ protons. The distances were statistically equivalent for all protons except C3-H and were consistent with the rotation within the active site or sliding parallel to the heme plane. When 50 microM alpha-NF was added, proton-heme iron distances ranged from 7.3 to 10.0 A. Consistent with kinetics of activation, the 1' position was situated closest to the heme, while the fluorophenyl 5-H proton was the furthest. Proton-heme iron distances for MDZ with CYP3A4 and 50 microM testosterone ranged from 7.7 to 9.0 A, with the flourophenyl 5-H proton furthest from the heme iron and the C4-H closest to the heme, also consistent with kinetic observations. When titrated with CYP3A4 in the presence of MDZ, testosterone and alpha-NF resonances themselves exhibited significant broadening and enhanced relaxation rates, indicating that these effector molecules were also bound within the CYP3A4 active site near the paramagnetic heme iron. These results suggest that the effector exerts its cooperative effects on MDZ metabolism through simultaneous binding of MDZ and effector near the CYP3A4 heme.