Molecular evidence for the expression of nicotinic acetylcholine receptor alpha-chain in mouse thymus.

The presence and structure of nicotinic acetylcholine receptor (nAChR) in the thymus has been a subject of interest for many years because of its possible role in the pathogenesis of the autoimmune disease myasthenia gravis. Using the polymerase chain reaction with primers specific for the alpha-chain of nAChR (nAChR-alpha), an 880-bp homologous band was found after amplification of cDNA prepared from mouse thymus, thymic medullary and cortical epithelial cell lines, but not from thymocytes or kidney. Sequencing of the polymerase chain reaction product from the thymus and thymic medullary and cortical epithelial lines showed identity with skeletal muscle nAChR-alpha over the region examined. This region includes the domains of the molecule on which B cell and T cell autoantigenic targets have been described. No evidence was found in mouse tissue for the exon 3A, which has been described in human muscle and the human rhabdomyosarcoma cell line TE671. Our results provide evidence at the RNA level for the expression of the nAChR-alpha on stromal cells but not on thymocytes in normal murine thymus and are consistent with a role for intrathymic autoantigen expression in the pathogenesis of myasthenia gravis.     

Biochemical effects of mild iron deficiency and cold acclimatization on rat skeletal muscle

Most of the previous studies on the effects of iron deficiency on skeletal muscle respiratory capacity and work performance have been investigated in severe or moderate iron-deficiency anemia. We report here that even in mild iron deficiency where the hemoglobin concentration was 10 g/dl and the iron stores in livers and spleen were not completely depleted, a marked reduction in succinate dehydrogenase was observed in skeletal muscles but not in heart. Similarly, cytochrome oxidase activities were reduced. Although no significant change in glycerophosphate dehydrogenase was detected in the iron-deficient rats, exposure to cold in this group greatly reduced this enzyme activity. As cold acclimatization accelerates marrow erythropoiesis (20) which in turn, demands more iron, it seems that in the iron-insufficient state, this iron demand for marrow activity may persist at the expense of the tissue iron pool, resulting in a marked reduction in glycerophosphate dehydrogenase activities. Since succinate dehydrogenase plays a significant role in the impairment of mitochondrial function and early fatigue of iron-deficient muscle (11), the present study shows that even in mild iron deficiency, some loss of muscle functions could result as succinate dehydrogenase activities were greatly reduced.     

Production of pro-opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by POMC-converting enzyme

Pro-opiomelanocortin (POMC) was expressed in CV-1 (green monkey kidney) cells using a vaccinia virus transient expression system [(1986) Proc. Natl. Acad. Sci. USA 83, 8122]. The system involved infection of cells with a recombinant vaccinia virus carrying the T7 RNA polymerase gene and transfection with a plasmid containing the mouse POMC sequence flanked by the T7 RNA polymerase promoter at its 5'-end and the T7 RNA polymerase terminator at its 3'-end. Assay of the medium from transfected cells showed that 1-2 micrograms of immunoreactive ACTH was produced/10(6) cells. Analysis of the same medium by SDS-PAGE/Western blots revealed a band of 30-36 kDa, which was immunostained with both ACTH and beta-endorphin antisera. Labeling the transfected cells with [3H]Arg, followed by immunoprecipitation and SDS-PAGE showed the synthesis of a major peak of POMC, 33 kDa. Purified [3H]POMC expressed by CV-1 cells was cleaved in vitro by bovine intermediate lobe secretory vesicle pro-opiomelanocortin-converting enzyme to ACTH intermediates (19-25 kDa), beta-lipotropin and beta-endorphin. Thus, this work has demonstrated a technique for expressing microgram quantities of prohormones in mammalian cells, suitable for use as substrates for prohormone-converting enzymes in vitro.     

Modulation of cortisol receptors in embryonic retina cells by changes in cell-cell contacts: Correlations with induction of glutamine synthetase

Cortisol induces glutamine synthetase (GS) in neural retina tissue of chick embryos. GS induction represents a characteristic feature of embryonic retina differentiation. However, if the tissue is dissociated into single cells, the dispersed cells are not inducible for GS. We report that cell dispersion results in a rapid and marked reduction in the level of cortisol-binding cytoplasmic receptors. This reduction persists if the cells are maintained in a dispersed state. However, if the cells are reaggregated and they reconstruct tissue-like contacts and architecture, the level of cortisol receptors increases, and so does inducibility for GS. The results indicate that, in the embryonic neural retina histotypic cell contacts and interactions are involved in regulating the level of cortisol receptors. We propose that cell contact-dependent signals from the cell surface may modulate levels of cytoplasmic cortisol receptors necessary for GS induction.     

NEUROSECRETORY CELL PROTEIN METABOLISM IN THE LAND SNAIL,OTALA LACTEA

—Protein synthesis in an identified molluscan neurosecretory cell of the land snail, Otala lactea was examined using three different types of polyacrylamide gel electrophoresis. Cells taken from active snails synthesized specific low molecular weight proteins while those from aestivated snails did not. Most of the newly synthesized low molecular weight proteins in the active snails were lost from the cell body when the preparations was chased for 19 h in label-free enriched medium in the presence of anisomycin, an inhibitor of protein synthesis. If colchicine, a blocker of axonal transport, was included in the chase medium, the proteins present following a pulse were largely replaced by smaller molecular weight species. The results suggest that specific low molecular weight proteins are converted to smaller species and then transported from the cell body.     

Chlormethiazole in treatment of status epilepticus.

Chlormethiazole (Heminevrin) was successful in controlling fits in seven out of nine episodes of intractable status epilepticus. It was administered as a constant intravenous injection at rates of up to 0.7g/h. No serious side effects were encountered, and the drug deserves wider recognition as a useful therapeutic agent in the management of status epilepticus.     

Location of a second partitioning region (ParL) on the F plasmid

Abstract The leading region of the F plasmid has been found to extend the maintenance of the normally unstable plasmid vector pACYC184. This ability is due to effective partitioning of plasmid molecules at cell division. Cloning, deletion analysis and transposon mutagenesis have located the partitioning region (ParL) to be encoded within 63.65–64.11F. Complementation studies indicated that parL is a cis -acting locus.     

Motor neuropathy‐associated mutation impairs Seipin functions in neurotransmission

Gain‐of‐toxic‐function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N‐glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N‐glycosylation of Seipin on synaptic transmission by over‐expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over‐expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post‐synaptic currents and miniature inhibitory post‐synaptic currents. The amplitude of both evoked excitatory post‐synaptic currents and inhibitory post‐synaptic current is also compromised by mutant Seipin over‐expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over‐expressing Seipin‐N88S, whereas neither γ‐amino butyric acid (GABA) nor α‐Amino‐3‐hydroxy‐5‐methyl‐4‐ isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin‐N88S‐expressing neurons. These data demonstrate that Seipin‐N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse.