Mechanism of Agonist-Induced Down-Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.     

Potent convulsant actions of the adenosine receptor antagonist, xanthine amine congener (XAC)

The convulsant properties of xanthine amine congener (XAC, 8-(4-(2-aminoethyl)-aminocarboxylmethyloxy)phenyl-1,3-dipropylxant hine) are compared to those of caffeine. Male Swiss albino mice were infused with convulsants through a lateral tail vein. Convulsion thresholds (i.e. the amount of convulsants required to elicit convulsions) of 39.8 +/- 2.0 mg/kg (n = 10) and 109.8 +/- 2.3 mg/kg (n = 10) were calculated for XAC and caffeine respectively. Pretreatment of animals with the adenosine receptor agonists 2-chloroadenosine, N6-cyclohexyladenosine or 5'-N-ethylcarboxamido-adenosine (1 mg/kg, i.p., 20 minutes prior to infusion) significantly decreased the seizure threshold of both XAC and caffeine. The adenosine uptake blockers, 6-nitrobenzylthioinosine or dipyridamole (0.25 mg/kg, i.p., 20 minutes prior to infusion) did not significantly affect the seizure threshold to either XAC or caffeine. The benzodiazepine agonist diazepam (5 mg/kg, i.p., 20 minutes prior to infusion) significantly increased the seizure threshold to both XAC (p less than 0.05) and caffeine (p less than 0.01), whereas the benzodiazepine antagonist Ro 15-1788 (10 mg/kg, i.p., 20 minutes prior to infusion) significantly increased the seizure threshold to caffeine (p less than 0.01), but not XAC. The results suggest that actions at benzodiazepine receptors may be a tenable hypothesis to explain the convulsant actions of caffeine, but not those of XAC.     

Neurofibromatosis type 2 appears to be a genetically homogeneous disease

Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome characterized by the development of vestibular schwannomas and other tumors of the nervous system, including cranial and spinal meningiomas, schwannomas, and ependymomas. The presence of bilateral vestibular schwannomas is sufficient for the diagnosis. Skin manifestations are less common than in neurofibromatosis type 1 (NF1; von Recklinghausen disease). The apparent clinical distinction between NF1 and NF2 has been confirmed at the level of the gene locus by linkage studies; the gene for NF1 maps to chromosome 17, whereas the gene for NF2 has been assigned (in a single family) to chromosome 22. To increase the precision of the genetic mapping of NF2 and to determine whether additional susceptibility loci exist, we have performed linkage analysis on 12 families with NF2 by using four polymorphic markers from chromosome 22 and a marker at the NF1 locus on chromosome 17. Our results confirm the assignment of the gene for NF2 to chromosome 22 and do not support the hypothesis of genetic heterogeneity. We believe that chromosome 22 markers can now be used for presymptomatic diagnosis in selected families. The NF2 gene is tightly linked to the D22S32 locus (maximum lod score 4.12; recombination fraction 0). A CA-repeat polymorphism at the CRYB2 locus was the most informative marker in our families (lod score 5.99), but because the observed recombination fraction between NF2 and CRYB2 was 10 cM, predictions using this marker will need to be interpreted with caution.     

Aminoglycoside-3″-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum

The bacterial gene aad A encodes the enzyme aminoglycoside-3-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aad A gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.     

Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly

One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3 genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the divergence of sequence between the amplified copies. Received: 22 November 1995 / Accepted: 23 February 1996     

An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus are able to repair and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. The ability of R1 to repair completely > 100 double-strand breaks in its chromosome without lethality or mutagenesis is recA dependent. However, during the first 1.5 h after irradiation, recA+ and recA cells show similar increases in the average size of chromosomal fragments. In recA+ cells, DNA continues to enlarge to wild-type size within 29 h. However, in recA cells, no DNA repair is observed following the first 1.5 h postirradiation. This recA-independent effect was studied further, using two slightly different Escherichia coli plasmids forming adjacent duplication insertions in the chromosome, providing repetitive sequences suitable for circularization by non-recA-dependent pathways following irradiation. After exposure to 1.75 Mrad (17,500 Gy), circular derivatives of the integration units were detected in both recA+ and recA cells. These DNA circles were formed in the first 1.5 h postirradiation, several hours before the onset of detectable recA-dependent homologous recombination. By comparison, D. radiodurans strains containing the same E. coli plasmids as nonrepetitive direct insertions did not form circular derivatives of the integration units before or after irradiation in recA+ or recA cells. The circular derivatives of the tandemly integrated plasmids were formed before the onset of recA-dependent repair and have structures consistent with the hypothesis that DNA repair occurring immediately postirradiation is by a recA-independent single-strand annealing reaction and may be a preparatory step for further DNA repair in wild-type D. radiodurans.     

Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations

The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system. Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity. Several mutantmerT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Glyl4Arg, Glyl5Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter.     

Determination of two sites of automethylation in bovine erythrocyte protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase - EDTA disodium ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl fluoride - TEA trifluoroacetic acid - HPLC high-pressure liquid chromatography     

A mathematical model for fibro-proliferative wound healing disorders

The normal process of dermal wound healing fails in some cases, due to fibro-proliferative disorders such as keloid and hypertrophic scars. These types of abnormal healing may be regarded as pathologically excessive responses to wounding in terms of fibroblastic cell profiles and their inflammatory growth-factor mediators. Biologically, these conditions are poorly understood and current medical treatments are thus unreliable. In this paper, the authors apply an existing deterministic mathematical model for fibroplasia and wound contraction in adult mammalian dermis (Olsenet al., J. theor. Biol. 177, 113–128, 1995) to investigate key clinical problems concerning these healing disorders. A caricature model is proposed which retains the fundamental cellular and chemical components of the full model, in order to analyse the spatiotemporal dynamics of the initiation, progression, cessation and regression of fibro-contractive diseases in relation to normal healing. This model accounts for fibroblastic cell migration, proliferation and death and growth-factor diffusion, production by cells and tissue removal/decay. Explicit results are obtained in terms of the model processes and parameters. The rate of cellular production of the chemical is shown to be critical to the development of a stable pathological state. Further, cessation and/or regression of the disease depend on appropriate spatiotemporally varying forms for this production rate, which can be understood in terms of the bistability of the normal dermal and pathological steady states—a central property of the model, which is evident from stability and bifurcation analyses. The work predicts novel, biologically realistic and testable pathogenic and control mechanisms, the understanding of which will lead toward more effective strategies for clinical therapy of fibro-proliferative disorders.     

Narratives of Jewish Identity

Sojourners: The Return of German Jews and the Question of Identity. John Borneman and Jeffrey M. Peck. Lincoln
Recovered Roots: Collective Memory and the Making of Israeli National Tradition. Yael Zerubavel. Chicago
The Masada Myth: Collective Memory and Mythmaking in Israel. Nachman Ben-Yehuda. Madison