New constraint on the existence of the µ+ → e+ γ decay.

The analysis of a combined data set, totaling 3.6 × 10(14) stopped muons on target, in the search for the lepton flavor violating decay µ(+) → e(+)γ is presented. The data collected by the MEG experiment at the Paul Scherrer Institut show no excess of events compared to background expectations and yield a new upper limit on the branching ratio of this decay of 5.7 × 10(-13) (90% confidence level). This represents a four times more stringent limit than the previous world best limit set by MEG.

New limit on the lepton-flavor-violating decay µ+→e+γ.

We present a new result based on an analysis of the data collected by the MEG detector at the Paul Scherrer Institut in 2009 and 2010, in search of the lepton-flavor-violating decay µ(+)e(+)γ. The likelihood analysis of the combined data sample, which corresponds to a total of 1.8×10(14) muon decays, gives a 90% C.L. upper limit of 2.4×10(-12) on the branching ratio of the µ(+)→e(+)γ decay, constituting the most stringent limit on the existence of this decay to date.

Expression of diverse Na+ channel messenger RNAs in rat myocardium. Evidence for a cardiac-specific Na+ channel.

This study examined the diversity of Na+ channel gene expression in intact cardiac tissue and purified myocardial cells. The screening of neonatal rat myocardial cell cDNA libraries with a conserved rat brain Na+ channel cDNA probe, resulted in the isolation and characterization of a putative rat cardiac Na+ channel cDNA probe (pCSC-1). The deduced amino acid sequence of pCSC-1 displayed a striking degree of homology with the eel, rat brain-1, and rat brain-2 Na+ channel, thereby identifying pCSC-1 as a related member of the family of Na+ channel genes. Northern blot analysis revealed the expression of a 7-kb CSC-1 transcript in rat cardiac tissue and purified myocardial cells, but little or no detectable expression of CSC-1 in rat brain, skeletal muscle, denervated skeletal muscle, or liver. Using RNase protection and Northern blot hybridization with specific rat brain Na+ channel gene probes, expression of the rat brain-1 Na+ channel was observed in rat myocardium, but no detectable expression of the rat brain-2 gene was found. This study provides evidence for the expression of diverse Na+ channel mRNAs in rat myocardium and presents the initial characterization of a new, related member of the family of Na+ channel genes, which appears to be expressed in a cardiac-specific manner.

Reduction of expression of the multidrug resistance protein (MRP) in human tumor cells by antisense phosphorothioate oligonucleotides.

Multidrug resistance protein (MRP) is a member of the ATP-binding cassette superfamily of transport proteins which has been demonstrated to cause multidrug resistance when transfected into previously sensitive cells. Sixteen eicosomeric oligonucleotides complementary to different regions along the entire length of the MRP mRNA reduced MRP mRNA and protein levels in drug-resistant small cell lung cancer cells that highly overexpress this protein. In MRP-transfected HeLa cells that express intermediate levels of MRP, one oligonucleotide, ISIS 7597, targeted to the coding region of the MRP mRNA, decreased the levels of MRP mRNA to < 10% of control levels in a concentration-dependent manner. This effect was rapid but transient with a return to control levels of MRP mRNA 72 hr after treatment. A double treatment with ISIS 7597 produced a sustained inhibition, resulting in a greater than 90% reduction in MRP mRNA for 72 hr and a comparable decrease in protein levels. Increased sensitivity to doxorubicin was observed under these conditions. Northern blotting analyses using two DNA probes corresponding to sequences 5' and 3' of the ISIS 7597 target sequence, respectively, revealed the presence of low levels of two smaller sized RNA fragments as expected from an RNase H-mediated mechanism of action of the antisense oligonucleotide. These studies indicate that a specific reduction in MRP expression can be achieved with antisense oligonucleotides, a finding that has potential implications for the treatment of drug-resistant tumors.

[Anomalous implantation of the appendiceal base: a clinical case]. / Anomalia di impianto della base appendicolare: caso clinico.

The authors report a case of anomalous implantation of the base of the appendix, observed in a 8-year-old boy, arising from the anterior wall of ascending colon at 15 cm from the ileo-cecal valve. After a brief review of the most frequent congenital anomalies of the appendix and the few cases of anomalous implantation described in international literature, the authors emphasize the importance of an accurate exploration and mobilization of the ascending colon to reduce the risk of diagnostic and therapeutic mistakes, also related to the small incision of McBurney, usually made for the routine laparotomic appendectomy.

Determination of synthesis rate and lifetime of bacterial mRNAs.

A method has been developed to determine the synthesis rate and lifetime of bacterial mRNAs, either bulk mRNA or specific mRNAs, with a minimum of physiological disturbance. The method uses hybridization of pulse-labeled RNA to specific probes followed by an evaluation based on a computer simulation of the labeling kinetics of different classes of RNA. The method was applied to the determination of bulk mRNA in Escherichia coli growing in glucose minimal medium: 60% of the instantaneous rate of RNA synthesis, or 2.3% of the total amount of RNA, was found to be mRNA with an average lifetime of 1.0 +/- 0.2 min (= 0.7 min half-life).

Stringent and growth control of rRNA synthesis in Escherichia coli are both mediated by ppGpp.

Weak stringent or relaxed responses were induced in Escherichia coli (relA+), using mild amino acid starvation or treatment with chloramphenicol at low concentrations, respectively, such that the growth rate was barely reduced. In this manner, the intracellular concentration of the nucleotide guanosine tetraphosphate, ppGpp, could be varied in any desired range between 0 and 1000 pmol of ppGpp per OD460 unit of culture mass. At the same time, the rate of synthesis of stable RNA (rs; rRNA and tRNA) was measured, relative to the total instantaneous rate of RNA synthesis (rt). The correlation between the cytoplasmic concentration of ppGpp and stable RNA gene activity (rs/rt) was the same as that observed previously with relA+ and relA strains growing exponentially at different rates in different media. This suggests that the distinction between growth control and stringent control of stable RNA synthesis is arbitrary, and that both kinds of control reflect the same ppGpp-dependent phenomenon. By increasing the stable RNA gene dosage, using high copy number plasmids carrying an rrn gene, we have tested the idea that ppGpp partitions the bacterial RNA polymerase into two forms with different probabilities to initiate at stable RNA and mRNA promoters. The relaxed response was not significantly altered, but the extent of the stringent response was reduced by the presence of extra rrn genes. The results agree with quantitative predictions derived from the RNA polymerase partitioning hypothesis.

Control of rRNA synthesis in Escherichia coli at increased rrn gene dosage. Role of guanosine tetraphosphate and ribosome feedback.

The effects of extra, plasmid-borne rRNA genes on the synthesis rate of rRNA in Escherichia coli were examined by measuring the fraction of total RNA synthesis that is rRNA and tRNA (rs/rt), the cytoplasmic concentration of guanosine tetraphosphate (ppGpp), and the absolute rates of RNA and protein synthesis. Experiments were carried out in different growth media and with two different strains of E. coli, B/r and K-12. The results indicated: 1) increased rrn gene dosage from either intact or defective rrn genes reduced bacterial growth rates and ribosome activity (protein synthesis rate/average ribosome), and increased rs/rt. 2) Extra intact, but not extra defective, plasmid-borne rrn genes caused the level of ppGpp to be increased in comparison to the pBR322-carrying control strain. 3) As a function of ppGpp, rs/rt was increased with either intact or defective rrn genes. 4) The rRNA synthesis rate/rrn gene was reduced in the presence of extra rrn genes; this reduction in gene activity was greater with intact than with defective rrn genes. An analysis of these results showed that they are consistent with the ppGpp hypothesis of rRNA control but not with a feedback effector role of translating ribosomes.

Studies in vivo on Escherichia coli RNA polymerase mutants altered in the stringent response.

Previous studies on two Escherichia coli rpoB mutants, carrying single amino acid substitutions at approximate amino acid positions 736 and 906 in the beta subunit, showed that these alterations in the RNA polymerase resulted in an apparent reduced response to valine-induced amino acid starvation in vivo and prevented ppGpp-mediated inhibition of transcriptional initiation at stable RNA promoters in vitro. These observations suggested that the mutations had altered either the ppGpp binding site or the promoter selectivity of the enzyme. The in vivo analysis presented here indicates that these mutants encode an RNA polymerase that responds normally to changes in the level of ppGpp; their apparent relaxedness is due to a reduced accumulation of ppGpp during isoleucine starvation. Thus, there is no indication that the mutations have altered ppGpp binding sites. These observations and the difference between in vitro and in vivo results can be explained by the assumption that the mutations produce an extended ppGpp-dependent pausing of RNA polymerase during the transcription of unstable RNA. Comparison of the vivo and in vitro effects of ppGpp on rrn transcription further suggests that these reflect different phenomena, although in both cases ppGpp inhibits rrn transcription.

Coupling muscle electrical activity to gene expression via a cAMP-dependent second messenger system.

Embryonic-type nicotinic acetylcholine receptor (nAChR) gene expression is regulated by muscle activity. The mechanism by which this activity is transduced to the genome is not known. We have addressed this issue by using a rat primary muscle cell culture system that mimics the in vivo effects of muscle activity on nAChR expression. We report here that the suppression of nAChR gene expression by muscle activity can be reversed by increasing intracellular cAMP levels. This effect is specific to the embryonic-type receptor genes. Electrically insensitive genes such as those encoding the adult-type nAChR epsilon-subunit and creatine kinase are not up-regulated by cAMP. In addition, muscle inactivity caused either by tetrodotoxin or denervation increases cAMP levels and protein kinase A activity, consistent with their proposed role in mediating nAChR gene expression. Finally, we report that this same mechanism appears to regulate other genes, such as those encoding the tetrodotoxin-insensitive sodium channel, MyoD, and myogenin which, like the nAChR, are regulated by muscle electrical activity. Based on these results it is proposed that muscle electrical activity is coupled to gene expression via a cAMP-dependent second messenger system.

An isoform variant of the cytomegalovirus immediate-early auto repressor functions as a transcriptional activator.

The major immediate-early promoter (MIEP) of human cytomegalovirus directs the expression of several differentially spliced and polyadenylated mRNAs. These mRNAs encode nuclear phosphorproteins (IE55, IE72, and IE86), which consist of common and unique amino acid sequences. To date, very little is known of the functional role of the 55-kDa (IE55) protein. Here we present evidence that the IE55 protein is a positive activator of the MIEP. In human fibroblast cells IE55 protein activated the MIEP between 10- and 30-fold. Fusion of IE55 to the GAL4 DNA binding domain resulted in a chimeric protein capable of trans-activating a reporter with GAL4 recognition sequences. These results strongly suggest that IE55 is a bona fide transcriptional activator protein. In addition, the IE55 protein was found not to act synergistically with the IE72 activator protein. The IE55 protein shares the same amino acid sequence as IE86 except for a 154-amino-acid deletion at the C-terminal end of the protein. These proteins were functionally antagonistic; IE55 relieved repression by IE86 and, conversely, IE86 negated IE55 activation. Mutagenesis of the MIEP revealed that the target sequence for activation by IE55 is different from the IE86 autorepressive response element. These experiments suggest that the mechanism of action of the IE55 and IE86 isoforms is distinct. Moreover, from these results it is apparent that the interplay of these factors might be critical in determining the level of HCMV replication in the host.

Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. Identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression.

To study the mechanisms which mediate the transcriptional activation of cardiac genes during alpha adrenergic stimulation, the present study examined the regulated expression of three cardiac genes, a ventricular embryonic gene (atrial natriuretic factor, ANF), a constitutively expressed contractile protein gene (cardiac MLC-2), and a cardiac sodium channel gene. alpha 1-Adrenergic stimulation activates the expression and release of ANF from neonatal ventricular cells. As assessed by RNase protection analyses, treatment with alpha-adrenergic agonists increases the steady-state levels of ANF mRNA by greater than 15-fold. However, a rat cardiac sodium channel gene mRNA is not induced, indicating that alpha-adrenergic stimulation does not lead to an increase in the expression of all cardiac genes. Studies employing a series of rat ANF luciferase and rat MLC-2 luciferase fusion genes identify 315- and 92-base pair cis regulatory sequences within an embryonic gene (ANF) and a constitutively expressed contractile protein gene (MLC-2), respectively, which mediate alpha-adrenergic-inducible gene expression. Transfection of various ANF luciferase reporters into neonatal rat ventricular cells demonstrated that upstream sequences which mediate tissue-specific expression (-3003 to -638) can be segregated from those responsible for inducibility. The lack of inducibility of a cardiac Na+ channel gene, and the segregation of ANF gene sequences which mediate cardiac specific from those which mediate inducible expression, provides further insight into the relationship between muscle-specific and inducible expression during cardiac myocyte hypertrophy. Based on these results, a testable model is proposed for the induction of embryonic cardiac genes and constitutively expressed contractile protein genes and the noninducibility of a subset of cardiac genes during alpha-adrenergic stimulation of neonatal rat ventricular cells.