A link between RNA interference and nonsense-mediated decay in Caenorhabditis elegans.

Double-stranded RNA (dsRNA) inhibits expression of homologous genes by a process involving messenger RNA degradation. To gain insight into the mechanism of degradation, we examined how RNA interference is affected by mutations in the smg genes, which are required for nonsense-mediated decay. For three of six smg genes tested, mutations resulted in animals that were initially silenced by dsRNA but then recovered; wild-type animals remained silenced. The levels of target messenger RNAs were restored during recovery, and RNA editing and degradation of the dsRNA were identical to those of the wild type. We suggest that persistence of RNA interference relies on a subset of smg genes.

Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA.

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in Caenorhabditis elegans. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.

Detection of inosine in messenger RNA by inosine-specific cleavage.

Double-stranded RNA adenosine deaminases catalyze the conversion of adenosine to inosine within double-stranded RNA. A few candidate biological substrates for these enzymes have been discovered by noticing discrepancies between genomic and cDNA sequences. Toward the goal of finding a systematic approach to identify new deaminase substrates, we developed a method to cleave RNA specifically after inosine and an amplification strategy to identify the cleavage sites. We tested our method on a candidate substrate, the messenger RNA for glutamate receptor subunit B (GluR-B). We detected cleavage of the endogenous GluR-B message from rat brain at two known RNA editing sites, thus providing the first direct evidence for the presence of inosine at these sites. The described method will facilitate the mapping of inosines within RNA and, most importantly, will provide a way to identify new deaminase substrates.

Suppression of loss-of-function mutations in Escherichia coli ribonuclease P RNA (M1 RNA) by a specific base-pair disruption.

A plasmid encoding ribonuclease P RNA of Escherichia coli (M1 RNA) was mutagenized with hydroxylamine in vitro and defective rnpB genes were identified by screening in an in vivo suppression assay. Defective rnpB sequences were mutagenized with a second round of hydroxylamine to restore activity. We report here that conversion of the C32.G48 base-pair of RNase P RNA to either C.A or U.G restored activity to defective rnpB genes bearing a variety of spatially distinct primary mutations. Disruption of this base-pair in an otherwise wild-type rnpB sequence increased the growth rate of the indicator strain E. coli FS101, consistent with the opening of C32.G48 during in vivo assembly of or catalysis by RNase P.

Sequences encoding the protein and RNA components of ribonuclease P from Streptomyces bikiniensis var. zorbonensis.

The genes encoding the RNA (rnpB) and protein (rnpA) subunits of ribonuclease P (RNase P) of Streptomyces bikiniensis var. zorbonensis have been cloned by complementing the temperature-sensitive growth phenotype of Escherichia coli strains that carry mutations in these genes. The rnpB sequence of S. bikiniensis includes new covariations that lead to refinement of the previous secondary structure models for RNase P RNAs. The deduced amino acid sequence of S. bikiniensis RNase P is conserved with that of other known RNase P proteins only to a limited extent. Immediately upstream from rnpA is an open reading frame that codes for the highly conserved ribosomal protein, L34. This same gene arrangement occurs in all bacteria studied to date.

Long-range structure in ribonuclease P RNA.

Phylogenetic-comparative and mutational analyses were used to elucidate the structure of the catalytically active RNA component of eubacterial ribonuclease P (RNase P). In addition to the refinement and extension of known structural elements, the analyses revealed a long-range interaction that results in a second pseudoknot in the RNA. This feature strongly constrains the three-dimensional structure of RNase P RNA near the active site. Some RNase P RNAs lack this structure but contain a unique, possibly compensating, structural domain. This suggests that different RNA structures located at different positions in the sequence may have equivalent architectural functions in RNase P RNA.

Pacemaker system failure and other events in children with surgically induced heart block.

Of 1,193 consecutive pediatric (less than 18 years) patients undergoing intracardiac repair from 1975 to 1984, 38 (3.2%) developed surgically induced complete heart block and were treated by permanent pacemaker implantation. Anomalies included complete atrioventricular septal defect = 9 (24%), simple ventricular septal defect = 9 (24%), atrioventricular discordant connection = 8 (212), tetralogy of Fallot = 7 (182), and other complex anomalies = 5 (13%). There were no hospital deaths. follow-up was 100% complete. There were six late deaths = 16%. Actuarial survival was 79 + 9% at 10 years. None of the late deaths were related to disturbance of cardiac rhythm or pacemaker system failure. Twelve patients (32%), required 27 reoperations for various types of pacemaker system failure. Indications for reoperation included: lead failure (44%). Pulse generator failure (44%), and wound sepsis (12%). Actuarial freedom from any pacemaker related reoperation was 50 + 16% at 48 months and 25 + 15% at 96 months. Only first reoperation was found to be an incremental risk factor for subsequent reoperation (p = 0.03). Surgical heart block has been neutralized as a risk factor for hospital death after repair of congenital cardiac defects. The risk of the development of surgical heart block now approaches zero, as indicated by a decreased incidence (1 of 401 = 0.25%) in our institution from 1985 to 1987, as compared to the era 1975 to 1984 (p = 0.001).

Chronotropic response of an activity detecting pacemaker compared with the normal sinus node.

We compared the rate responsiveness of an activity-detecting multiprogrammable, single chamber pacemaker (Medtronic Activitrax) to rate responsiveness of the normal sinus node. This pacemaker changes its basic pacing rate in response to physical activity. The rate responsiveness is programmable by selecting one of three activity thresholds, and one of 10 rate response settings. The study included a group of six normal volunteers and 12 patients implanted with Activitrax to examine the similarity of the pacemaker rate to normal sinus rhythm during acceleration and deceleration. The pacemaker was set to Activity mode, at a basic rate of 60 bpm. In volunteers, the device was externally secured on the chest wall and tested at two programmed settings. When programmed at a high threshold of activity and high rate response in volunteers, there was no significant difference in maximum normal sinus rates and pacemaker rates during arm waving, jumping in place, and walking during stress testing. At a medium activity threshold, the only significant difference occurred during submaximal stress testing, when the maximum sinus rate achieved was 141 +/- 19 bpm and the maximum pacing rate was 105 +/- 8 bpm (p less than .02). The pacemaker behaved in a similar manner in patients, successfully simulating the typical fast acceleration and slow deceleration of a normal sinus node in exercise testing. There was no difference in pacer response when implanted in abdominal or infraclavicular locations. The implanted units have functioned normally over a follow-up period of nine to 22 months. Activitrax can be programmed to achieve physiologic pacing rates in response to normal daily activities with appropriate programming.

Transtelephonic monitoring of 25,919 implanted pacemakers.

In order to evaluate the behavior of lithium-powered cardiac pacemakers, a database of 25,919 lithium-powered pacing systems including 23,517 single and 2,402 dual chambered pacemaker generators were followed in 21,750 patients. Of this group, 11,319 were currently active in addition to 7,560 who died, as well as 2,871 patients who terminated their follow-up service prior to the end-of-life of the pacemaker system. A total of 23,517 single chambered pacemakers were followed for 719,173 months of pacing. The mean time to explant for generator malfunctions was 42 months and for all pacing system malfunctions it was 38 months compared to 31.9 months and 28.6 months, respectively, for the 2,402 dual chambered units that were observed for 38,718 months. In the single chambered units, the most frequent reason for explant was battery exhaustion (37% of explants) followed by lead problems (26%) compared to 49% and 12%, respectively, for the dual chambered units. The incidence rates, defined as a transtelephonic test result, required physician decision for action or clarification of the pacing mode or program parameters and showed a high incidence rate immediately post-implant; thereafter, there was a relatively trouble-free period until the 36th month when capture, sensing, and battery problems began to occur at a steady rate reaching 4% at the end of 48 months. Similarly, for dual chambered units, an 8% incidence rate was seen within the first 3 months, followed by a quiescent period until the 28th month when the incidence rate jumped to 16%.(ABSTRACT TRUNCATED AT 250 WORDS)

Esophageal P-synchronous pacing.

An esophageal electrode can be employed to provide atrial sensing which then can be used to change from temporary right ventricular (VVI) pacing to P-synchronous (VAT) pacing. Two cases of postoperative aortic valve replacement, each with new complete heart block (CHB) and dopamine dependency, are presented. In both cases, establishment of P-synchronous pacing resulted in improved hemodynamic status characterized by successful weaning from dopamine and maintenance of adequate cardiac output (CO).

Redundant pacing: case report and review.

A 17-year-old boy with severe sick sinus bradycardia had a doubly redundant pacemaker inserted. With two separate ventricular leads inserted by the epicardial route, the pacemaker is able to compensate for a lead that has developed high threshold by activation of an alternate lead. The two pacing channels of the pulse generator can be programmed independently of one another. In addition, there is a back-up pacing circuit separate from the two primary channels. The pacer can be used with both channels active for continuous automatic redundancy for safety, or with one channel active and the other in reserve.

Fatty acid oxidation intermediates and the effect of fasting on oxidation in red and white skeletal muscle.

In vitro oxidation of U-[14C]glucose-6-phosphate, 2-[14C]pyruvate, and 1-[14C]pyruvate was significantly reduced in red skeletal muscle from fasting rats. Over the same time interval of fasting, 1-[14C]palmitate oxidation remained unchanged. Pyruvate dehydrogenase activity, assayed before in vitro activation, was also reduced in red muscle. Long chain acylcarnitine and long chain acyl CoA increased over the same time period in red muscle. Changes in long chain fatty acid derivatives and pyruvate oxidation were much less dramatic in white muscle. We propose that the rise in levels of long chain fatty acid derivatives may be directly related to the inhibition of carbohydrate oxidation during fasting in red skeletal muscle.

Skeletal muscle fatty acid oxidation during early postnatal development in the rat.

(1-14C)-palmitate oxidation in rat skeletal muscle homogenates increased only minimally from birth until 15 days of age and then increased more than five times between 15 days and adulthood. In contrast, liver oxidation of palmitate reached adult levels by 4 days of age. Although muscle carnitine concentration, carnitine palmityl transferase activity, and total muscle protein increased during the neonatal period, these changes did not completely parallel the rise in muscle palmitate oxidation. Palmityl-CoA synthetase and palmityl-CoA dehydrogenase activities also did not parallel the rise in overall palmitate oxidation.

Massive mitral regrgitation from chordal rupture and coronary artery disease.

The precise mechanism that causes spontaneous rupture of chordae tendineae remains unknown. That it may occur in patients with no disease other than underlying or associated coronary artery occlusion has not been previously reported. Six patients with chordal rupture were found among 600 patients who underwent operation for mitral regurgitation in a 6-year period. All 6 patients without exception underwent simultaneous mitral valve replacement and coronary revascularization. The salient clinical features of these patients are summarized, and 1 case is reported in detail.