Protein-tyrosine phosphatases specifically regulate muscle adult-type nicotinic acetylcholine receptor gene expression

Innervation of skeletal muscles results in expression of adult-type nicotinic acetylcholine receptors (alpha 2 beta epsilon delta) beneath the neuromuscular junction. This local expression is largely a result of selective induction of adult-type nicotinic acetylcholine receptor (nAChR) genes in endplate-associated myonuclei. The molecular mechanism by which the nerve induces gene expression in these nuclei is not known. We have shown previously that ionophore-induced calcium influx across the plasma membrane preferentially decreases expression from the adult-type specific nAChR epsilon-subunit gene (Walke, W., Staple, J., Adams, L., Gnegy, M., Chahine, K., and Goldman, D. (1994) J. Biol. Chem. 269, 19447-19456). Here we provide evidence that the genes encoding adult-type nAChRs are specifically regulated by protein-tyrosine phosphatase activity. Orthovanadate, a specific protein-tyrosine phosphatase inhibitor, caused increased expression of the epsilon-subunit gene in rat primary myotubes and was able to completely block the suppressive effects of increased calcium influx on epsilon-subunit RNA expression. Overexpression of protein-tyrosine phosphatases selectively decreased expression from the adult-type nAChR genes with no effect on the embryonic-type specific gamma-subunit gene. These results demonstrate that protein-tyrosine phosphatases regulate mammalian adult-type nAChR gene expression and suggest a mechanism by which muscle innervation selectively regulates gene expression in endplate-associated myonuclei.     

Identification of a poxvirus gene encoding a uracil DNA glycosylase

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.     

Structure of the m1 muscarinic acetylcholine receptor gene and its promoter

The m1 receptor is one of five muscarinic receptors that mediate the metabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia. RNase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire m1 gene demonstrated that the rat m1 gene consists of a single 657-base pairs (bp) non-coding exon separated by a 13. 5-kilobase (kb) intron from a 2.54-kb coding exon that contains the entire open reading frame. The splice acceptor for the coding exon starting at -71 bp relative to the adenine of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940 and Wood, I. C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221-14225). Like the m4 gene, the m1 promoter lacks TATA and CAAT consensus motifs, and the first exon and 5'-flanking region are not gc-rich. The 5'-flanking region also contains the consensus regulatory elements Sp-1, NZF-1, AP-1, AP-2, E-box, NFkappaB, and Oct-1. Unike the m4 promoter, there is no evidence of a RE1/NRSE silencer element in the m1 promoter. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence and the first exon are sufficient to drive cell-specific expression of reporter gene in IMR32 neuroblastoma cells while remaining silent in 3T3 fibrobasts.     

Identification of a new cis-regulatory element in a Nicotiana tabacum polyubiquitin gene promoter

With the development of powerful computer systems, computer-assisted medical diagnosis and therapy have become common over the last 10 years. Even in the surgical field, computer- and robotic-assisted techniques are becoming practical but are not yet used on a daily basis. In the orthopaedic field, computer and robotic assistance is used in planning and performing demanding three-dimensional osteotomies, setting pedicle screws in the spine and milling the femoral medullary canal in total hip replacement. This article introduces a computer- and robotic-assisted system for performing arthroplasty in total knee replacement procedures.     

Developmental expression of a DNA repair gene in Arabidopsis

As the use of electroconvulsive therapy (ECT) increases, the chance of a practitioner's encountering a patient with significant heart failure, ventricular dysfunction, or valvular heart disease also increases. This article reviews the epidemiology, pathophysiology, and available data on the risk of ECT in these patients. Recommendations are made regarding evaluation and treatment of such patients. Some special situations are identified that may require a modification of routine procedures. Overall, ECT can be performed safely in most patients with underlying cardiac conditions, as long as appropriate precautions are taken to identify these patients ahead of time.