Ribosomal heterogeneity from chromatin diminution in Ascaris lumbricoides.

The genome of Ascaris lumbricoides encodes both germline- and soma-specific proteins homologous to the eukaryotic small ribosomal protein (Rp) S19. The two Ascaris homologs differ by 24 amino acid substitutions and are both components of the small ribosomal subunits. In oocytes, the germline RpS19 homolog (RpS19G) predominates. During chromatin diminution, however, the gene is eliminated from all presomatic cells, and RpS19G is replaced by the product of the somatic gene (RpS19S). Chromatin diminution in A. lumbricoides causes a change in the protein composition of ribosomes during development and represents an alternative means of gene regulation.

Chromatin diminution in nematode development.

Chromatin diminution in Parascaris and Ascaris represents the classical case of a developmentally programmed genome rearrangement. The process is very specific with respect to ontogenetic timing and chromosomal localization, and involves chromosomal breakage, new telomere formation and DNA degradation. Recent evidence from Ascaris lumbricoides var. suum suggests that chromatin diminution might have a function in gene regulation.

Developmentally regulated telomerase activity is correlated with chromosomal healing during chromatin diminution in Ascaris suum.

Telomerase is the ribonucleoprotein complex responsible for the maintenance of the physical ends, or telomeres, of most eukaryotic chromosomes. In this study, telomerase activity has been identified in cell extracts from the nematode Ascaris suum. This parasitic nematode is particularly suited as a model system for the study of telomerase, because it shows the phenomenon of chromatin diminution, consisting of developmentally programmed chromosomal breakage, DNA elimination, and new telomere formation. In vitro, the A. suum telomerase is capable of efficiently recognizing and elongating nontelomeric primers with nematode-specific telomere repeats by using limited homology at the 3' end of the DNA to anneal with the putative telomerase RNA template. The activity of this enzyme is developmentally regulated, and it correlates temporally with the phenomenon of chromatin diminution. It is up-regulated during the first two rounds of embryonic cell divisions, to reach a peak in 4-cell-stage embryos, when three presomatic blastomeres prepare for chromatin diminution. The activity remains high until the beginning of gastrulation, when the last of the presomatic cells undergoes chromatin diminution, and then constantly decreases during further development. In summary, our data strongly argue for a role of this enzyme in chromosome healing during the process of chromatin diminution.

A newly formed telomere in Ascaris suum does not exert a telomere position effect on a nearby gene.

During the process of chromatin diminution in Ascaris suum (formerly named Ascaris lumbricoides var. suum), developmentally regulated chromosomal fragmentation and new telomere addition occur within specific chromosomal breakage regions (CBRs). The DNA sequences flanking one of these CBRs (CBR-1) were analyzed, and two protein-encoding genes were found on either side. The noneliminated gene, agp-1, whose AUG start codon is located within approximately 2 kb of the boundary of CBR-1, encodes a putative GTP-binding protein which is structurally related to eukaryotic and prokaryotic elongation factors. Northern (RNA) blot analyses revealed that transcripts of this gene are present at all developmental stages, suggesting that the massive chromosomal rearrangements associated with the process of chromatin diminution have no influence on agp-1 expression. This demonstrates that addition of new telomeres in CBR-1 does not result in a telomeric position effect, a phenomenon previously described in Saccharomyces cerevisiae.

Quantitative analysis of RNA, glycogen and nucleotides from different developmental stages of Ascaris lumbricoides (var. suum).

Cold acid phenol extractions (Kirby, K.S. (1968) in Methods in Enzymology, Vol. XII B, pp. 87--99, Academic Press, New York) of homogenates from oocytes, zygotes and larval stages of Ascaris lumbricoides yield predominantly glycogen, some glycogen . phosphate complexes and guanine nucleotides, but only small amounts of RNA. Oocytes contain 10.0 pg of extractable 19 S and 26 S rRNA and 1.1 pg 4 S and 5 S RNA per cell. During the maturation of the oocyte to the zygote stage, the amount of 19 S and 26 S rRNA increases by 57%, and that of 4 S and 5 S RNA by 130%. Larval stages contain 62% more 19 S and 26 S rRNA, and 47% more 4 S and 5 S RNA than zygotes. The amount of the extractable glycogen decreases by about 80% and nucleotides by about 70% from oogenesis to the larval stage. The Kirby extract from homogenates of spermatids contains only traces of glycogen, glycogen . phosphate complexes and nucleotides, and 115 times less RNA per spermatid than oocytes. The nucleotide pool sizes of oocytes, zygotes and larval stages were determined; the pools consist of 71--88% guanine nucleotides. Spermatids contain only adenine nucleotides and an unidentified, nucleotide-like compound.

Intracavitary electrode catheter cardioversion of atrial tachyarrhythmias in the dog.

This study examined factors determining efficacy of intracavitary cardioversion of atrial tachyarrhythmias in closed chest, anesthetized dogs with talc pericarditis. Electrode catheters were positioned transvenously with the cathode in the right atrial appendage. In Group 1 dogs (n = 6), three anode sites (superior and inferior venae cavae ostia and mid-right atrium) were tested with graded energy shocks to determine the lowest effective cardioversion energy at each anode position. In Group 2 dogs (n = 9), multiple cardioversion attempts with energy levels of 0.01 to 5.0 J were used to evaluate reproducibility of energy thresholds. In Group 3 dogs (n = 6) without talc-induced pericarditis, atrial pathologic study was done after five intracavitary shocks (0.5 or 5.0 J). In Group 1, cardioversion was achieved with 0.75 J or less with no significant difference in minimal effective cardioversion energies among the three anode positions tested. In Group 2, 98 (26%) of 372 cardioversion attempts were successful. Intra-animal minimal effective cardioversion energies varied widely, and timing of shocks relative to atrial electrograms did not influence efficacy. Complications were infrequent and included delayed sinus rhythm recovery, transient atrioventricular block and ventricular fibrillation. Ventricular fibrillation occurred in 9 (2.4%) of 372 shocks, and was associated with higher delivered energies (6 of 9 with greater than or equal to 1.0 J) and with shocks delivered 116 to 180 ms after onset of the QRS complex. In Group 3, two dogs had no histologic damage, three dogs had multiple small foci of subendocardial necrosis and in one dog these foci coalesced to involve half the atrial wall thickness. Thus, low energy cardioversion of atrial tachyarrhythmias is feasible using intracavitary electrodes. Synchronization of energy delivery to the QRS complex is important to minimize risk of ventricular fibrillation.

Bromocriptine treatment of digitalis-induced ventricular tachyarrhythmias: studies in a canine model.

Ventricular tachyarrhythmias associated with digitalis toxicity are believed to be due, in part, to cardiac glycoside-mediated increased central sympathetic neural activity. Because dopaminergic receptor agonists reduce sympathetic outflow, this study assessed effectiveness of the available dopaminergic agonist, bromocriptine, in slowing or terminating ouabain-induced ventricular tachycardia in anesthetized dogs. In all experiments, ouabain was administered intravenously (20 micrograms/kg body weight bolus injection, followed by 2.5 micrograms/kg per min infusion) until the onset of stable ventricular tachycardia. Of seven untreated dogs (Group 1), ouabain-induced ventricular tachyarrhythmias resulted in ventricular fibrillation in three, while in four dogs tachycardia persisted without significant change in rate until the study was terminated. Fourteen dogs (Group 2) received bromocriptine, either 30 micrograms/kg (Group 2A) or 50 micrograms/kg (Group 2B), after the onset of ventricular tachycardia. Tachycardia slowed in all 14 dogs and terminated with resumption of sinus rhythm in 8 of the 14. In all six dogs pretreated with the peripheral dopaminergic antagonist domperidone (Group 3), bromocriptine, 50 micrograms/kg, slowed ventricular tachycardia and in three of the six, tachycardia terminated. In contrast, of five dogs pretreated with haloperidol, a central and peripheral dopaminergic receptor antagonist (Group 4), bromocriptine, 50 micrograms/kg, failed to slow ventricular tachycardia in three, and two of the three developed ventricular fibrillation. In summary, the dopaminergic receptor agonist, bromocriptine, presumably acting at central dopaminergic receptor sites, consistently slowed and in most cases reversed ouabain-induced ventricular tachycardia in a canine model.

Two highly reiterated nucleotide sequences in the low C-value genome of Panagrellus redivivus.

Two families of highly reiterated satellite nucleotide (nt) sequences have been found in the genome of the sexually separated nematode Panagrellus redivivus. The repeats are arranged in tandem arrays but the different satellites are not intermingled. Monomeric lengths are of 155 bp for one kind and 167 bp for the other; they were named E155 and E167. The A + T content is elevated in both families (i.e., 59.5%, and 65.3%, respectively). No similarity was found between the two satellites nor to other known highly repetitive elements. Furthermore, nt methylation as well as transcriptional activity were negative. An internal subrepeating unit, about 30 bp long, was observed in E167, implying that it could have evolved from a shorter sequence. Reiteration frequencies are approx. 30,000 and 40,000 copies per haploid genome, for E155 and E167, respectively, constituting together about 17% of the total DNA. This figure is astonishingly high, considering a C-value of 70,000 kb in P. redivivus, which is thought to be the lower limit for metazoans. Hence, the genome complexity is approx. 58,000 kb. In contrast to the nematodes Ascaris lumbricoides and Parascaris equorum, however, P. redivivus does not seem to eliminate large blocks of satellite DNA in the presomatic cells during early development.

Tas, a retrotransposon from the parasitic nematode Ascaris lumbricoides.

The cloned retrotransposon Tas OE3 from the genome of the parasitic nematode Ascaris lumbricoides was completely sequenced. The element is flanked by long terminal repeats (LTR) and contains three distinct regions encoding putative proteins typical for retroid elements. The first region, ORF1, encodes a putative Gag protein including a 'Leu zipper', a nucleic acid binding motif, as well as an aspartic protease domain. The second region contains an incomplete ORF (ORF2) with sequence similarities to known retroviral reverse transcriptases (RT), ribonucleases H and integrases. A third ORF, which is located adjacent to the 3' LTR, might encode an env-like protein. Based on amino-acid sequence analysis of the RT domain, Tas falls into a new subgroup of LTR-containing retrotransposons.

Cloning and expression in vitro of a gene encoding tRNAArgACG from the nematode Caenorhabditis elegans.

A gene (rtr-1) coding for the tRNAArgACG has been isolated and characterized from the nematode, Caenorhabditis elegans. The coding portion is not interrupted by an intron and is followed by a track of four thymidines associated with termination by RNA polymerase III. The predicted mature product is 76 nucleotides (nt) long including the CCA tail, and is specific for the most used Arg codon in C. elegans. The gene can be transcribed and processed in a homologous in vitro system. The 82-nt primary transcript begins at the first purine upstream from the mature tRNA 5' end and terminates after the first thymidine of the terminator signal.

Stereological changes in the capillary network and nerve cells of the aging human brain.

Stereologic parameters of the capillaries and nerve cells of the brain cortex and putamen were investigated. Thirty-eight brains from subjects aged between 19 and 94 years were examined. All cases were free of metabolic, neurologic and psychiatric diseases. It is demonstrated that the capillary diameter remains unchanged during aging in both brain cortex and putamen. However, in the putamen the total capillary length per unit volume and the capillary volume fractions increase (approximately 60%) progressively with age. Consequently the mean inter-capillary distances in the putamen decrease (approximately 15%). These age-induced changes in the putamen indicate shrinking of subcortical brain structures. In contrast to those of the putamen, the morphometric data of the capillaries in the cortex remain unchanged during the aging process. Stereologic investigations of nerve cells in the brain cortex and putamen revealed that only in brains over 85 years of age can a significant decrease in nerve cell size be demonstrated. A correlation of all the data by a correspondence analytical procedure showed that only the surface/volume ratio of the capillaries correlates with the nerve cell size. This observation suggests a functional interaction between the nerve cells and the capillaries. From the data presented it becomes apparent that the shrinkage of the gyri in the aging brain is not a change in the volume of the cortex, but a decrease in the volume of subcortical structures.

Role of extrastimulus site and tachycardia cycle length in inducibility of atrial preexcitation by premature ventricular stimulation during reciprocating tachycardia.

Preexcitation of the atria during reciprocating tachycardia (RT) by a premature ventricular complex occurring when the His bundle is refractory provides direct evidence of the presence of accessory atrioventricular (AV) connection. The impact of ventricular stimulation site and RT cycle length on inducibility of atrial preexcitation was assessed in 38 patients with RT utilizing a single accessory AV connection (right free wall in 5 patients, left free wall in 21 and posterior septal/paraseptal in 12). Extrastimuli were inserted at right ventricular (RV) apical, left ventricular (LV) septal and LV free wall sites. Inducibility of and magnitude of atrial preexcitation increased as stimulation site approached accessory AV connection site. Thus, for RV free wall connections, RV extrastimuli preexcited the atria in 5 of 5 patients, LV septal in 1 of 5 and LV free wall in 0 of 4. For LV free wall accessory connections, RV extrastimuli preexcited the atria in only 3 of 21 patients, compared with 12 of 17 with LV septal and 20 of 21 with LV free wall stimulation. Additionally, the magnitude of atrial preexcitation achieved was related to RT cycle length, diminishing as cycle length shortened. Finally, in a few instances both RV apical and LV free wall extrastimuli failed to elicit preexcitation in patients with a posterior septal connection. Thus, ventricular pacing site and RT cycle length contribute importantly to induction of atrial preexcitation by ventricular extrastimulation technique and should be considered during evaluation of patients with RT in whom accessory AV connections may be present.

Electrophysiologic effects of flecainide acetate and its major metabolites in the canine heart.

Flecainide acetate, an investigational class 1 antiarrhythmic agent, undergoes biotransformation in man with production of 2 major metabolites: meta-O-dealkylated flecainide (S-24623) and the meta-O-dealkylated lactam of flecainide (S-26191). This study compared the effects of flecainide, S-24623 and S-26191 on cardiac electrophysiologic characteristics in the anesthetized dog. Each dog received 2 dose levels of 1 of the 3 test compounds after control measurements. Flecainide (2 and 4 mg/kg in 8 dogs), S-24623 (4 and 8 mg/kg in 8 dogs) and S-26191 (4 and 10 mg/kg in 7 dogs) were administered intravenously in dilute solution. Of the 3 compounds, only flecainide significantly prolonged sinus cycle length (p less than 0.01). However, both flecainide and S-24623 significantly prolonged minimum atrial paced cycle length with 1:1 atrioventricular conduction, atrioventricular nodal effective and functional refractory periods, and right ventricular effective refractory period. Metabolite S-26191 exhibited qualitatively similar but much weaker electrophysiologic actions. The maximal electrophysiologic effects of flecainide and S-24623 were approximately equivalent, but the metabolite was about one-half as potent on a milligram-permilligram basis, and lacked marked effects on infranodal (HV interval) conduction. S-26191 was less than one-tenth as potent as flecainide. Therefore, since both flecainide metabolites occur primarily in the conjugated form in plasma (i.e., free metabolite concentrations are low), it is unlikely that these compounds either potentiate flecainide's antiarrhythmic action or increase susceptibility to drug toxicity in the clinical setting.

Chromatin diminution in the parasitic nematodes ascaris suum and parascaris univalens.

Chromatin diminution in Parascaris univalens and Ascaris suum undoubtedly represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. It is a complex mechanism involving chromosomal breakage, new telomere addition and DNA degradation, and occurs in all presomatic cells. The process is rather specific with respect to its developmental timing and the chromosomal regions that are eliminated. The functional significance of chromatin diminution still remains an enigma. The fact, however, that single-copy, protein-coding genes are contained in the eliminated DNA demonstrates that in P. univalens and A. suum, there is a qualitative difference between germ-line and somatic genomes, and suggests that chromatin diminution may be used as a "throw-away" approach to gene regulation. We present a hypothesis as to how, during evolution, a partial genome duplication might have been linked to the process of chromatin diminution, in order to provide a selective advantage to parasitic DNA-eliminating nematodes.

Frequency and location of atherosclerotic plaques in the ascending aorta.

Among 97 specimens of ascending aorta from adults with clinical coronary disease, the prevalence of atherosclerotic plaques greater than 8 mm in diameter was 38%. The right side of the ascending aorta was more commonly involved than the left; the sites least commonly involved were the right-posterior, upper right-anterior, and lower posterior locations. Of specimens with plaques at the orifice of the innominate artery, 80% also had plaques in the ascending aorta, and 73% of specimens with plaques at the orifice of the left subclavian artery had plaques in the ascending aorta.

Clinical experience with the Novacor ventricular assist system. Bridge to transplantation and the transition to permanent application.

At Stanford University, a Novacor left ventricular assist system (Baxter Healthcare Corporation, Novacor Division, Oakland, Calif.) was placed as a bridge to heart transplantation in 13 patients. During the hospitalization preceding device implantation, all patients were receiving inotropic support for biventricular failure, 11 had pulmonary edema, 6 had life-threatening ventricular arrhythmias, 5 had liver dysfunction with coagulopathy, and 2 had renal failure necessitating artificial support. The mean cardiac index before implantation of the Novacor system was 1.5. All survivors with the Novacor device had a dramatic increase in cardiac output (mean cardiac index = 3.1). One patient with cardiac allograft rejection died during implantation of the left ventricular assist system. Two patients died of pulmonary sepsis and multiorgan failure after the device was implanted. All patients who had the Novacor device implanted for more than 7 days were able to walk and ride stationary bicycles while awaiting transplantation. Ten patients (77%) underwent successful heart transplantation after a mean of 18 days' support with the Novacor device. One patient died of presumed sepsis 2 days after transplantation. Nine patients (90%) are alive 4 months to 6 years after transplantation. In the overall United States experience, 68 patients (as of May 1990) have had a Novacor left ventricular assist device implanted. Five were still being supported, 39 had received a transplant (62%), and 35 patients (90%) survived the transplant hospitalization (1 died later). No instances of device failure have occurred. Overall, the Novacor assist system provided effective bridging to transplantation, with posttransplant survival similar to results after routine transplantation. Modifications and improvements based on this clinical experience have been made in the areas of patient selection, techniques of operative placement, postoperative management, and design of the assist system. Isolated left heart support with a fully implantable left ventricular assist system will be offered as an alternative to heart transplantation for selected patients by 1992.

Electrophysiologic properties of the myocardial infarction border zone: effects of transient aortic occlusion.

This study used transmural multipolar electrodes, ventricular pressure monitoring, and cardiac electrical stimulation techniques to examine the effects of transient aortic occlusion on ventricular refractoriness in a canine model of recent myocardial infarction. Six previously instrumented resting awake dogs were atrially paced, followed by timed premature extrastimuli inserted at epicardial pacing sites adjacent to an apical left ventricular (LV) myocardial infarction or in an LV control zone remote from the myocardial infarction. Electrophysiologic and pressure recordings were obtained before and during periods of transient aortic occlusion. Aortic occlusion was applied before the last beat of an eight-beat atrial pacing sequence and resulted in increased peak LV pressure (92.8 +/- 27.7 mm Hg, p = 0.003). Aortic occlusion shortened LV effective refractory period (ERP) recorded from the myocardial infarction border zone in both the subepicardial (-17.0 +/- 11.8 msec, p = 0.019) and subendocardial (-17.7 +/- 10.9 msec, p = 0.011) regions, whereas LVERP of the control zone was unchanged. Conduction latency of premature beats at equivalent coupling intervals and maximum latency observed were unchanged by aortic occlusion. atrioventricular conduction interval shortened in association with aortic occlusion. Thus transient aortic occlusion reduced ventricular refractoriness in the border zone adjacent to the myocardial infarction while control zone refractoriness was minimally or not changed. Heterogeneity of ventricular myocardial refractoriness may result from mechanical dysfunction, potentially increasing susceptibility to arrhythmias.

Isoenzyme specific inhibition of the reactivation of in vitro dissociated lactic dehydrogenase isoenzymes by two different peptides isolated from human liver.

The catalytic activity of the LDH-isoenzymes depends on their tetrameric structure. Low pH or other denaturants leads to dissociation into monomers and to the loss of the specific activity. After removal of the denaturing conditions reassociation and reactivation occur spontaneously. Neither NADH nor NAD+ shows a significant effect on the reactivation. We have isolated two different peptides which isoenzyme specifically inhibit the reactivation of dissociated LDH. Inhibition was abolished by treating with proteases. Additionally, NAD+ and NADH were found to be antagonists of the inhibitors. The heart-type enzyme-inhibitor system is especially susceptible for NADH whereas NAD+ affects the inhibition only slightly. The muscle-type system shows the opposite behavior, e.g., the completely inhibited system can be fully reactivated by NAD+ but not by NADH. These findings together with first kinetic studies suggest a possible specific regulatory function of these peptides.

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and -H4) isozymes.

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375--1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1.1.27) isoenzymes (LDH-M4 = muscle type; LDH-H4 = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M4 or LDH-H4. From the amino acid analyses, gel filtrations and PAGE + SDS, molecular weights of 1800 for the M4 and approximately 2700 for the H4 inhibitor were calculated. An apparent Ki of approximately 3 X 10(-5) mM for the H4 and approximately 7 X 10(-5) mM for the H4 inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M4-specific) and 2.4 (LDH-H4-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides react with monomers, dimers or a transition state during the tetramerisation process. kappa 1 for the dimerisation step of M4 = 2.0 X 10(5) M-1 . S-1 and of H4 = 8.2 X 10(4) M-1 . S-1; kappa 2 for the tetramerisation step of M4 = 2.8 X 10(5) M-1 . S-1 and of H4 = 1.2 X 10(5) . M-1 S-1, were calculated, the second step still being the faster one (Rudolf, R. and Jaenicke, R. (1976) Eur. J Biochem. 63, 409--417).

Thromboembolism in patients undergoing thoracotomy.

To determine the incidence of thromboembolism in relation to thoracotomy, 77 patients undergoing pulmonary resection were prospectively studied up to 30 days postoperatively for deep venous thrombosis and pulmonary embolism. Overall, 20 of 77 patients (26%) had thromboembolic events during their hospitalization. Four deep venous thromboses and 1 pulmonary embolism were detected in 5 of 77 patients preoperatively for an incidence of 6%. Postoperative thromboembolism was detected in 15 of 77 (19%): deep venous thrombosis in 11 (14%) and pulmonary embolism in 4 (5%). No postoperative thromboembolisms occurred in the 17 patients receiving preoperative aspirin or ibuprofen, whereas they did occur in 25% of the remainder (15/60). Thromboembolism after pulmonary resection was more frequent with bronchogenic carcinoma than with metastatic cancer or benign disease (15/59 [25%] versus 0/18 [0%]; p < 0.01), adenocarcinoma compared with other types of carcinoma (11/25 [44%] versus 4/34 [12%]; p < 0.0004), large primary lung cancer (> 3 cm in diameter) compared with smaller lesions (9/19 [47%] versus 6/40 [15%]; p < 0.0001), stage II compared with stage I (7/14 [50%] versus 7/34 [21%]; p < 0.04), and pneumonectomy or lobectomy compared with segmentectomy and wedge resection (14/49 [29%] versus 1/28 [4%]; p < 0.005). Three of 4 patients with thromboembolism detected preoperatively had operation within the previous year. Postoperative pulmonary embolism was fatal in 1 of 4 (25%) and accounted for the one death. These results suggest patients undergoing thoracotomy for lung cancer, especially adenocarcinoma, should be considered for thromboembolic prophylaxis.