Differential time course of angiotensin-induced AP-1 and Krox proteins in the rat lamina terminalis and hypothalamus

Binding to olfactory receptors is the first step in odorant and pheromonal recognition and discrimination. These receptors constitute one of the most important, although poorly known, families of neuronal receptors. In this study we used degenerated oligonucleotides and a RT-PCR approach to selectively amplify olfactory receptors in the nasal epithelium of the domestic pig Sus scrofa. Several combinations of oligonucleotide were tested and allowed the isolation of eleven different partial sequences belonging to the seven transmembrane olfactory receptor family. These receptors formed a separate family within the seven transmembrane receptor superfamily in pigs. Using the criteria of Ben Arie et al. [Ben-Arie N., Lancet D., Taylor C., Khen M., Walker N., Ledbetter DH., Carrozzo R., Patel K., Sheer D., Lehrah H. and North M., Hum. Mol. Genet., 3 (1994) 229-235], the 11 receptors described here can be classified into three known families and seven subfamilies (one known and six new).     

Mono- and dysfunctional nitrogen mustard analogues of the DNA minor groove binder pibenzimol. Synthesis, cytotoxicity and interaction with DNA

Two series of mono- and dysfunctional aniline mustards linked to a bisbenzimidazole minor groove binder have been prepared using a new method (polyphosphate ester-mediated direct coupling of appropriate mustard acids with a preformed advanced phenylenediamine intermediate). As the linker chain attaching the mustard was lengthened the binding site size of the compounds to calf thymus DNA remained essentially constant at 2.6 nucleotides, but reversible binding strength declined by a factor of 2. Analogues with longer linker chains alkylated DNA much more rapidly than those with shorter chains, consistent with the electronic factors. The short chain analogues also failed to alkylate a 120 bp HindIII to Bg/II fragment of the gpt gene, as measured by gel electrophoresis cleavage assays. The longer chain analogues (both mono- and dysfunctional mustards) showed patterns of DNA alkylation that varied with chain length. In particular, while most compounds showed substantial N7 alkylation at many guanine residues, the analogue with a (CH2)3 linker chain showed strong alkylation at adenine sites in poly-AT regions. For the longer chain analogues, the bifunctional mustards were substantially (10- to 20-fold) more cytotoxic than the corresponding monofunctional analogues.     

Cochlear implantation. Practical information for the generalist

The diagnosis and investigation of a patient with bilateral, severe-to-profound sensorineural hearing loss is presented. The steps toward cochlear implantation, including tests of audition, balance, radiographic imaging, and the pre- and postrehabilitation period, are reviewed. The two major cochlear implants available today, and future developments within the cochlear implant, are presented.     

Negative regulation of Wee1 expression and Cdc2 phosphorylation during p53-mediated growth arrest and apoptosis

The G2 cell cycle checkpoint protects cells from potentially lethal mitotic entry after DNA damage. This checkpoint involves inhibitory phosphorylation of Cdc2 at the tyrosine-15 (Y15) position, mediated in part by the Wee1 protein kinase. Recent evidence suggests that p53 may accelerate mitotic entry after DNA damage and that the override of the G2 checkpoint may play a role in the induction of apoptosis by p53. To determine the biochemical mechanism by which p53 inactivates the G2 checkpoint, the effects of p53 activation on Wee1 expression, Cdc2-Y15 phosphorylation, and cyclin B1-associated Cdc2 kinase activity were examined. Under conditions of either growth arrest or apoptosis, p53 activation resulted in the down-regulation of Wee1 expression and dephosphorylation of Cdc2. A parallel increase in cyclin B1/Cdc2 kinase activity was observed during p53-mediated apoptosis. Negative regulation of the Wee1 expression and Cdc2 phosphorylation by p53 was also evident in thymus tissue from p53+/+ mice but not from p53-/- mice. Inactivation of the G2 checkpoint may contribute to the tumor suppressor activity of p53.     

CTL response to Mycobacterium tuberculosis: identification of an immunogenic epitope in the 19-kDa lipoprotein

The successful resolution of infection with Mycobacterium tuberculosis (M.tb) is believed to involve the induction of CTLs that are capable of killing cells harboring this pathogen, although little information is known about the MHC restriction or fine specificity of such CTLs. In this study, we used knowledge of the HLA-A*0201-binding motif and an immunofluorescence-based peptide-binding assay to screen for potential HLA-A*0201-binding epitopes contained in the 19-kDa lipoprotein of M.tb (M.tb19). CD8+ T cells derived from HLA-A*0201+ patients with active tuberculosis (TB) as well as tuberculin skin test-positive individuals who had no history of TB were used as effector cells to determine whether these epitopes are recognized by in vivo-primed CTLs. An in vitro vaccination system using HLA-A*0201+ dendritic cells (DCs) as APCs was used to determine whether these epitopes can sensitize naive CD8+ T cells in vitro, leading to the generation of Ag-specific CTLs. The results show that an HLA-A*0201-binding peptide comprised of residues 88 to 97 of M.tb19 (P88-97) is recognized by circulating CD8+ CTLs from both healthy tuberculin skin test-positive individuals and patients with active TB but not by tuberculin skin test-negative subjects. Moreover, dendritic cells pulsed with this peptide induced class I MHC-restricted CTLs from the T cells of healthy unsensitized persons. Finally, CTL lines that were specific for P88-97 were shown to lyse autologous monocytes that had been infected acutely with the H37Ra strain of M.tb. These results demonstrate that M.tb19 elicits HLA class I-restricted CTLs in vitro and in vivo that recognize endogenously processed Ag. Epitopes of the type identified here may prove useful in the design of an M.tb vaccine.     

Stenting of "unprotected" left main coronary artery stenoses: early and late results

The effect of bone plug length and Kurosaka screw (DePuy, Warsaw, IN) diameter on graft holding strength of the bone-tendon-bone construct was determined. Random length porcine bone plugs were assigned to fixation with 7 or 9 mm Kurosaka screws. Peak load to failure was determined. There was a significant decrease in peak load to failure of the 5-mm long bone plugs compared with longer bone plugs. No difference was found between longer lengths of bone plug in either the 7- or 9-mm screw diameter groups. The 9-mm diameter screws significantly increased peak load to failure for both 1- and 2-cm bone plug lengths.     

The measurement of bioreductive capacity of tumor cells using methylene blue

PURPOSE: Methylene blue (MB) can be used as an intracellular electron acceptor. The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell suspensions. METHODS AND MATERIALS: We measured oxygen consumption by Clark electrode and pentose cycle activity by release of 14CO2 from 1-14C-glucose. RESULTS: Methylene blue catalyzes the reaction of intracellular reductants NADPH, NADH, and reduced glutathione (GSH) with oxygen, causing the production of hydrogen peroxide. The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction. In a cellular system MB causes the electron flow from cellular endogenous substrates to oxygen. It is activated by the disruption of the NADP+/NADPH ratio due to several processes. These are direct oxidation of NADPH and GSH, the GSH peroxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidation by oxidized glutathione (GSSG). This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cycle (PC) in the presence of MB. The cellular effect of MB differs from other electron accepting drugs. Diamide and tert-butylhydroperoxide act as direct oxidants, while MB is an electron carrier to oxygen. Accordingly, MB shows the highest effect on PC activation and oxygen consumption. CONCLUSIONS: Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measured by oxygen consumption and PC activation.     

Three-dimensional solution structure of alpha-conotoxin MII by NMR spectroscopy: effects of solution environment on helicity

alpha-Conotoxin MII, a 16-residue polypeptide from the venom of the piscivorous cone snail Conus magus, is a potent and highly specific blocker of mammalian neuronal nicotinic acetylcholine receptors composed of alpha3 beta2 subunits. The role of this receptor type in the modulation of neurotransmitter release and its relevance to the problems of addiction and psychosis emphasize the importance of a structural understanding of the mode of interaction of MII with the alpha3 beta2 interface. Here we describe the three-dimensional solution structure of MII determined using 2D 1H NMR spectroscopy. Structural restraints consisting of 376 interproton distances inferred from NOEs and 12 dihedral restraints derived from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. The final set of 20 structures is exceptionally well-defined with mean pairwise rms differences over the whole molecule of 0.07 A for the backbone atoms and 0.34 A for all heavy atoms. MII adopts a compact structure incorporating a central segment of alpha-helix and beta-turns at the N- and C-termini. The molecule is stabilized by two disulfide bonds, which provide cross-links between the N-terminus and both the middle and C-terminus of the structure. The susceptibility of the structure to conformational change was examined using several different solvent conditions. While the global fold of MII remains the same, the structure is stabilized in a more hydrophobic environment provided by the addition of acetonitrile or trifluoroethanol to the aqueous solution. The distribution of amino acid side chains in MII creates distinct hydrophobic and polar patches on its surface that may be important for the specific interaction with the alpha3beta2 neuronal nAChR. A comparison of the structure of MII with other neuronal-specific alpha-conotoxins provides insights into their mode of interaction with these receptors.     

Leukemia inhibitory factor modulates interleukin-1beta-induced activation of the hypothalamo-pituitary-adrenal axis

We have shown that leukemia inhibitory factor (LIF) is expressed in corticotroph cells and stimulates POMC gene expression and ACTH secretion in vivo and in vitro. We therefore examined the regulation of in vitro and in vivo pituitary LIF expression by cytokines known to stimulate the hypothalamo-pituitary-adrenal axis. In the corticotroph cell line AtT-20/D16v-F2, recombinant murine interleukin-1beta (IL-1beta; 0.1-10.0 ng/ml) caused a 5- to 10-fold increase in LIF messenger RNA (mRNA) levels. LIF mRNA expression was induced as early as 1 h, peaked at 2 h, and still persistently elevated above the baseline after 8 h. This effect of IL-1beta on LIF mRNA expression was abolished by preincubation with human IL-1 receptor antagonist (100 ng/ml) or antimurine IL-1beta antibody (10 microg/ml). Tumor necrosis factor-alpha (20 ng/ml) only modestly increased LIF mRNA, but was synergistic with IL-1beta (up to 2.5-fold). In contrast, IL-2 and IL-6 did not alter LIF mRNA. In C57BL/6 mice, i.p. injection of 100 ng IL-1beta increased plasma ACTH and corticosterone levels after 1 h (P < 0.02). In addition, pituitary LIF mRNA content was increased for up to 2 h in response to IL-1beta. In comparison to wild-type (+/+) B6D2F1 mice, LIF knockout mice with a deleted LIF gene (-/-) exhibited decreased plasma ACTH (631 +/- 61 vs. 376 +/- 50 pg/ml; P < 0.01) and corticosterone (783 +/- 85 vs. 433 +/- 51 ng/ml; P < 0.01) levels 1 h after i.p. IL-1beta administration. In conclusion, corticotroph LIF mRNA expression is specifically stimulated by IL-1beta and tumor necrosis factor-alpha. The attenuated hypothalamo-pituitary-adrenal response to IL-1beta in LIF knockout mice indicates that the effect of IL-1beta on ACTH secretion is modulated by LIF. Thus, LIF appears to function as an immune-neuroendocrine modulator signaling the hypothalamo-pituitary-adrenal axis.