Artificially ambiguous genetic code confers growth yield advantage.

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway.

Aminoacyl transfer RNA (tRNA) synthetases establish the rules of the genetic code by catalyzing the aminoacylation of tRNAs. For some synthetases, accuracy depends critically on an editing function at a site distinct from the aminoacylation site. Mutants of Escherichia coli that incorrectly charge tRNA(Val) with cysteine were selected after random mutagenesis of the whole chromosome. All mutations obtained were located in the editing site of valyl-tRNA synthetase. More than 20% of the valine in cellular proteins from such an editing mutant organism could be replaced with the noncanonical aminobutyrate, sterically similar to cysteine. Thus, the editing function may have played a central role in restricting the genetic code to 20 amino acids. Disabling this editing function offers a powerful approach for diversifying the chemical composition of proteins and for emulating evolutionary stages of ambiguous translation.

Results of aortic valve replacement with pulmonary and aortic homografts.

BACKGROUND AND AIM OF THE STUDY: Aortic valve replacement with cryopreserved human pulmonary or aortic valves (homografts) is an attractive alternative to the implantation of mechanical valves or bioprostheses, as anticoagulation can be avoided and a near-normal anatomy restored. However, few reports exist on the long-term follow up of patients with this type of valve. METHODS: Between 1990 and 1997, a total of 64 homografts were implanted in 62 adults (mean age 42 +/- 12 years) with non-endocarditic valve lesions (insufficiency, n = 16; stenosis, n = 20; combined lesions, n = 12; redo, n = 16). In total, 23 pulmonary grafts (PG) and 41 aortic grafts (AG) were used. Valves were obtained from the European Homograft Bank in Brussels. Two patients with aortic homografts were lost to follow up; the others were examined clinically and echocardiographically at yearly intervals (mean 3.6 +/- 2.0 years). Children aged less than 16 years (n = 21), and patients receiving a homograft due to endocarditis (n = 28) or during a Ross procedure (n = 16) were excluded from the study. RESULTS: Three patients (5%) died due to early postoperative complications (two with AG, one with PG). Three PG had to be explanted due to primary malfunction, and five (total 35%) during further follow up due to severe aortic insufficiency (at a mean of 3.3 +/- 1.8 years). In contrast, all AG were functioning at the end of the observation period (log rank test, p = 0.0001, chi-square test 13.9). The mean echocardiographic degree of regurgitation for PG was significantly higher than for AG (2.2 +/- 1 vs. 0.75 +/- 0.7, p <0.0001). The peak transvalvular gradient did not differ between groups (PG 12.3 +/- 9 mmHg vs. AG 16.7 +/- 10 mmHg, p = NS). In respect of perioperative parameters, patients with PG showed a significantly higher body temperature during the first seven postoperative days (37.3 +/- 0.6 degrees C vs. 36.8 +/- 0.3 degrees C, p = 0.003). All three patients with acute graft malfunction in long-term follow up had a perioperative febrile response without overt bacterial infection. CONCLUSION: In contrast to grafts of aortic origin, pulmonary homograft valves should not be used for aortic valve replacement because of their high rate of malfunction, both acutely and chronically. Higher postoperative body temperatures should lead to further investigations of possible enhanced immunoreactions against pulmonary homografts.

[Aortic valve replacement with homografts. An overview]. / Aortenklappenersatz mit Homografts. Eine Ubersicht.

The implantation of fresh or cryopreserved human heart valves (homografts) in aortic position is a tool in cardiac surgery since 30 years. Homografts are attractive alternatives to the implantation of mechanical or xenobiological prostheses, because anticoagulation can be avoided and a near normal anatomy can be restored. Physicians should know about the several kinds of grafts and operative techniques to adequately take care of the patients in follow-up. This overview on the literature covers methods of harvesting, preparation and conservation of homografts according to standard protocols of the European Homograft Bank in Brussels. Their use in the therapy of human valvular disease is discussed with special emphasis to operative techniques (subcoronary, root) and the Ross procedure and in pediatric surgery. Complications and aspects of postoperative care are discussed including immunologic phenomena. Homografts are useful tools for aortic valve replacement, especially in juveniles, in the presence of contraindications for anticoagulation and in endocarditis. Whereas aortic homografts have excellent long-term results, pulmonic homografts show a significant rate of malfunction. Further studies should be performed to clarify the role of the Ross operation or stentless xenografts compared to homografts in aortic position. In pediatric cardiac surgery homografts are of value especially for the reconstruction of the right ventricular outflow tract. Homografts in mitral position show disappointing results up to now. The major limitation in the use of homografts is the mismatch of availability and request, therefore homografts can only be used for the above mentioned special indications.

[Heart transplantation in myocardial sarcoidosis. Studies on the explanted heart]. / Herztransplantation bei Herzsarkoidose. Untersuchungen am explantierten Herzen.

HISTORY AND CLINICAL FINDINGS: A 40-year-old man with histologically proven sarcoidosis, known for 15 years, which had involved the myocardium was hospitalized because of intractable heart failure (NYHA class IV). An implantation of an intracardiac defibrillator for ventricular arrhythmias (Lown type IVa) had preceded. On physical examination severe dyspnea at rest cough and fever were noted. INVESTIGATIONS: The erythrocyte sedimentation rate (88/104 mm), C-reactive protein (250 mg/l) and white cell count (20/nl) were markedly increased. Serum sodium (113 mmol/l), potassium (3.0 mmol/l) and chloride (64 mmol/l) were markedly reduced, while creatinine (2.5 mg/dl) and urea (82 mg/dl) were elevated due to renal failure. The chest radiogram demonstrated central venous congestion, cardiomegaly and right pericardial infiltration. There were no obvious changes due to sarcoidosis and computed tomography did not indicate pulmonary involvement by sarcoidosis. The echocardiogram revealed severe impairment of left ventricular function with an ejection fraction of ca. 14%. DIAGNOSIS, TREATMENT AND COURSE: Heart failure (NYHA class IV), caused by a dilated cardiomyopathy of sarcoidosis, was accompanied by pneumonia which responded to antibiotics. But the chronic heart failure failed to improve on drug treatment and cardiac transplantation was undertaken. The explanted myocardium was examined histologically, immunologically and virologically. Antibodies were demonstrated against vascular endothelium, sarcolemma and endocardium (IgG and IgA), but not by PCR against cytomegalovirus, enterovirus and adenovirus. The transplantation was without complication and, under azathioprine and methylprednisolone, one rejection had occurred until now. The patient has been working full-time since 2 years in his previous occupation of lorry driver. There has been no evidence of renewed sarcoidosis activity. CONCLUSION: Sarcoidosis may involve the myocardium in up to 25% of cases. Clinically relevant symptoms are even more rare. Sarcoidosis should be included in the differential diagnosis of unexplained serious arrhythmias or cardiomyopathy, particularly in young persons. Cardiac transplantation may have to be contemplated if drug or pacemaker treatment fails to control heart failure.

Reassigning cysteine in the genetic code of Escherichia coli.

We investigated directed deviations from the universal genetic code. Mutant tRNAs that incorporate cysteine at positions corresponding to the isoleucine AUU, AUC, and AUA and methionine AUG codons were introduced in Escherichia coli K12. Missense mutations at the cysteine catalytic site of thymidylate synthase were systematically crossed with synthetic suppressor tRNACys genes coexpressed from compatible plasmids. Strains harboring complementary codon/anticodon associations could be stably propagated as thymidine prototrophs. A plasmid-encoded tRNACys reading the codon AUA persisted for more than 500 generations in a strain requiring its suppressor activity for thymidylate biosynthesis, but was eliminated from a strain not requiring it. Cysteine miscoding at the codon AUA was also enforced in the active site of amidase, an enzyme found in Helicobacter pylori and not present in wild-type E. coli. Propagating the amidase missense mutation in E. coli with an aliphatic amide as nitrogen source required the overproduction of Cys-tRNA synthetase together with the complementary suppressor tRNACys. The toxicity of cysteine miscoding was low in all our strains. The small size and amphiphilic character of this amino acid may render it acceptable as a replacement at most protein positions and thus apt to overcome the steric and polar constraints that limit evolution of the genetic code.

Long-term hemodynamic benefit of atrial synchronization with A2A2D or A2A2T pacing in sinus node syndrome after orthotopic heart transplantation.

BACKGROUND: Exercise capacity after heart transplantation (HTx) may be limited by sinus node disease of the donor heart and atrioatrial dissociation. The role of pacemaker therapy in this setting is not well defined. The purpose of this study was to compare clinical and hemodynamic data of heart transplant recipients with acquired sinus node disease treated with atrial synchronized pacing and patients with other pacing modes or without pacemakers 1 year after operation. METHODS: Our cohort comprises a total of 112 HTx recipients from the years 1984 to 1996. Atrial synchronized pacing was performed in 21 patients with donor sinus node disease and recipient sinus rhythm. There was no associated morbidity or death for the pacemaker implantation. Fourteen patients received a dual-chamber pacemaker programmed with a short atrioventricular-Delay in A2A2D mode (donor atrial pacing triggered by recipient atrial sensing or both atria stimulated on demand); in the last 6 consecutive patients a single-chamber pacemaker was implanted with two unipolar leads to the atria connected with a Y adapter programmed in A2A2T mode (both atria were sensed and stimulated by triggering each other). RESULTS: Signals and thresholds remain stable over time. When clinical and hemodynamic data of 12 A2A2D/T patients with complete 1 year follow-up were compared to age- and sex-matched control HTx recipients with other pacing modes or without pacemakers, a significant benefit of atrial synchronization could be shown regarding rise in heart rate response to exercise (+38% vs 30% vs 16% at 50 watt), New York Heart Association classification (1.6 vs 1.8 vs 2.2), Roskamm staging (1.3 vs 2.5 vs 1.5), cardiac index at rest (3.2 vs 2.78 vs 3.1 L/min x m2), cardiac index at 50 watt (5.5 vs 4.5 vs 5.2 L/min x m2), stroke work at rest (51 vs 38 vs 42 pondmeter [PM]), stroke work at 50 watt (66 vs 48 vs 51 PM), pulmonary wedge pressure at rest (7 vs 13 vs 8 mm Hg) and pulmonary wedge pressure at 50 watt (14 vs 24 vs 18 mm Hg). CONCLUSION: It is concluded that electromechanical synchronization of the atria was of long-term benefit in heart transplant recipients with recipient sinus rhythm and donor sinus node disease.

Atrial flap anastomosis: an alternative technique for orthotopic heart transplantation.

To combine the advantages of the standard technique and the bicaval technique of orthotopic heart transplantation, we use a muscular flap of recipient heart right atrium for connecting the superior vena cava with the donor heart right atrium. The results in respect to the maintenance of atrioventricular valve competence as well as atrial conduction are promising.

Successful thrombolysis after prosthetic pulmonary valve obstruction under aspirin monotherapy.

In a 22-year-old woman with recent onset of left-sided chest pain and exertional dyspnea, echocardiography revealed obstruction of a St. Jude Medical bileaflet prosthetic valve (size 23 mm) in the pulmonary position. Oral anticoagulation had been replaced for the previous 7 years by aspirin as the sole antithrombotic treatment. The valve had been inserted 16 years ago for pulmonary atresia. Valve function was restored by systemic application of 9 million units of urokinase.

[Primary chylopericardium--stepwise diagnosis and therapy of a differential diagnostically important illness]. / Primäres Chyloperikard--Stufendiagnostik und -therapie einer differentialdiagnostisch wichtigen Erkrankung.

Primary chylopericardium is a rare disease with a highly variable clinical course. We report on a 24-year old female with chylopericardium detected during a pulmonary infection. Despite successful treatment of the infectious disease, the chylopericardium persisted and led to cardiac tamponade. From this case, as well as from the literature, it is intriguing to postulate an inflammatory injury of preexisting anomalous lymphatic vessels leading to onset or aggravation of primary chylopericardium. The clinical hallmark of chylopericardium is a milky white, but odorless pericardial fluid at pericardiocentesis. For cases where conservative treatment and pericardiocentesis fail, we newly introduced the method of pericardio-peritoneal shunting by a pericardial window. With postoperative reaccumulation of pericardial fluid, total parenteral nutrition followed by medium chain triglyceride diet was successfully reinitiated. This combined surgical and conservative approach was performed for the first time and may have helped to avoid the more aggressive treatment of thoracic duct ligation and resection. During 2 years of follow-up the patient was asymptomatic and had no recurrence of pericardial effusion.

The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes.

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.

Changes in cardiac beta-adrenoceptors in human heart diseases: relationship to the degree of heart failure and further evidence for etiology-related regulation of beta 1 and beta 2 subtypes.

Radioligand binding studies were performed to investigate total beta-adrenoceptor density (Bmax) and beta 1 and beta 2 subtype distribution in left ventricular biopsies obtained from 8 prospective transplant donors serving as controls and from 143 patients with different degrees of heart failure (NYHA class II to IV) undergoing aortic or mitral valve surgery due to aortic or mitral stenosis, aortic or mitral regurgitation, as well as combined aortic or mitral valve lesions (stenosis and regurgitation). In 13 other patients, heart failure was due to hypertrophic obstructive cardiomyopathy (N = 6, NYHA III), tetralogy of Fallot (N = 4, NYHA III), or Becker's muscular dystrophy (N = 3, NYHA IV). Bmax was assessed by (-)-(125I)-iodocyanopindolol used as radioligand. Competition experiments with the highly selective beta 1-adrenoceptor antagonist CGP 20712A were performed for determination of beta 1- and beta 2-adrenoceptor subtypes. In biopsies taken from transplant donors, the Bmax was found to be 70.1 +/- 5.8 fmol/mg protein. In all groups investigated the extent of total beta-adrenoceptor downregulation was related to the degree of heart failure. The decrease in Bmax was found to be about 20% (NYHA II), 45% (NYHA III), and 60% (NYHA IV) when compared with controls. There was no significant difference in the reduction of total beta-adrenoceptor density between isolated aortic or mitral valve diseases and combined valve lesions. Independent of the degree of heart failure, selective downregulation of the beta 1 subtype was found in patients with isolated or combined aortic valve diseases, hypertrophic obstructive cardiomyopathy, and Becker's muscular dystrophy.(ABSTRACT TRUNCATED AT 250 WORDS)

[Successful operation of right ventricular cardiac fibroma in an infant]. / Erfolgreiche Operation eines rechtsventrikulären kardialen Fibroms bei einem Säugling.

Cardiac tumors are rare in infants. We describe the diagnostic procedure and the good postoperative outcome despite the very large cardiac fibroma.

Increased messenger RNA level of the inhibitory G protein alpha subunit Gi alpha-2 in human end-stage heart failure.

In human heart failure the positive inotropic and cAMP-elevating effects of both beta-adrenoceptor agonists and phosphodiesterase inhibitors are diminished. This has been explained at least in part by an increase in the inhibitory signal-transducing G protein (Gi) and unchanged stimulatory G protein (Gs). In the present study we determined the mRNA expression pattern of the alpha subunits of Gi-1, Gi-2, Gi-3, and Gs in myocardial tissue samples of patients undergoing heart transplantation. Northern blot analysis of total RNA extracted from left ventricles with 32P-labeled cDNAs demonstrated expression of Gi alpha-2, Gi alpha-3, and Gs alpha mRNA. In contrast, Gi alpha-1 mRNA was not detectable. To investigate whether the increased ratio of Gi/Gs might be due to altered gene expression, we compared mRNA levels of Gi alpha-2, Gi alpha-3, and Gs alpha in left ventricular myocardium from failing hearts with idiopathic dilated cardiomyopathy (n = 8) and ischemic cardiomyopathy (n = 6) and from nonfailing hearts from transplant donors (n = 8). Compared with nonfailing control hearts, the Gi alpha-2 mRNA was increased by 75 +/- 26% (p less than 0.05) in idiopathic dilated cardiomyopathy hearts and 90 +/- 26% (p less than 0.05) in ischemic cardiomyopathy hearts. Gi alpha-3 and Gs alpha mRNA levels were similar in the three groups. The results suggest that as in other mammalian species, Gi alpha-2 and Gi alpha-3 mRNA are the predominant Gi alpha mRNA subtypes in human ventricular myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional distribution of beta 1- and beta 2-adrenoceptors in the failing and nonfailing human heart.

Total beta-adrenoceptor density and beta 1- and beta 2-subtype distribution in right and left atria and in different ventricular regions from 14 failing and seven nonfailing human hearts have been compared. End-stage heart failure was due to idiopathic dilated cardiomyopathy (n = 8) or ischaemic cardiomyopathy (n = 6). In nonfailing hearts the total beta-adrenoceptor density was similar in the right and left atria and in all the ventricular regions studied (about 70 to 80 fmol/mg protein). The beta 1:beta 2-adrenoceptor ratio in both nonfailing atria was similar (about 70:30%) and was significantly smaller than in the different regions of both ventricles (about 80:20%). The beta 1-subtype density was similar in nonfailing atria and ventricles (about 55 fmol/mg protein). The beta 2-subtype density was significantly higher in the right and left atrium (about 25 fmol/mg protein) than in both ventricles (about 15 fmol/mg protein). In patients with end-stage heart failure due to idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy the total beta-adrenoceptor density was reduced by 50-60% in all regions. On the other hand, the beta 1- and beta 2-subtype distribution differed with the cause of heart failure. In patients with idiopathic dilated cardiomyopathy, the beta 1-adrenoceptor density was not significantly reduced. In patients with ischaemic cardiomyopathy both beta 1- and beta 2-adrenoceptors were reduced in all regions. It is concluded that downregulation of beta-adrenoceptors in patients with end-stage idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy occurs uniformly throughout the heart. The results support the hypothesis that changes in beta-adrenoceptor subtypes may be related to the cause of heart failure.

Dictyostelium annexin VII (synexin). cDNA sequence and isolation of a gene disruption mutant.

By cloning the cDNA coding for the membrane associated actin-binding protein p24, we identified a repetitive sequence motif consisting of the amino acids Gly, Tyr, Pro, Gln which is characteristic for a gene family in Dictyostelium discoideum. Using a cDNA probe corresponding to this motif, we isolated cDNA clones coding for a protein of the annexin family. On the basis of a long NH2-terminal sequence encompassing the Gly/Tyr/Pro/Gln motifs, the Dictyostelium annexin was identified as a homolog of vertebrate annexin VII (synexin). The mRNA coding for the Dictyostelium annexin VII has a size of 1.6 kilobases and is present during all developmental stages. Annexin VII is coded for by a single gene in Dictyostelium. A mutant deficient in annexin VII was isolated using a vector which carried the amino-terminal third of the Dictyostelium annexin VII cDNA followed by a viral epitope specific for a monoclonal antibody and a stop codon. Using this approach, homologous recombination in the annexin VII gene led to an expression of the viral epitope under the control of the endogenous annexin VII promoter. Lack of annexin VII is not a lethal event for D. discoideum, and the cells are able to undergo development on agar plates.

Gene structure of nuclear lamin LIII of Xenopus laevis; a model for the evolution of IF proteins from a lamin-like ancestor.

The lamin LIII gene of Xenopus laevis has been characterized. The gene is duplicated in the Xenopus genome. The transcribed region spreads over 22 kb of genomic DNA encoding 12 exons. Two alternatively spliced mRNAs are observed which encode LIII isoforms that differ only by the 12 C-terminal amino acids which, however, both contain the CaaX motif known to be the target of post-translational modifications. The intron pattern of the lamin LIII gene is strikingly similar to that of an invertebrate intermediate filament (IF) gene over the entire protein coding sequence. The similarity in gene structure is restricted to the rod domain when compared with vertebrate types I-III IF genes. Our data suggest a model of how IF proteins evolved from a lamin-like ancestor by deletion of two signal sequences; the nuclear localization signal and the C-terminal ras-related CaaX motif. The data rule out the previously proposed hypothesis that IF proteins evolved from an intronless ancestor with an early divergence of neuronal and non-neuronal IF proteins. Together with the data presented in the accompanying paper by Dodemond et al. it can be concluded that the tail domains of lamins and invertebrate IF proteins, but not those of vertebrate IF proteins, are homologous. Thus, the different vertebrate IF proteins probably evolved by combination of the central rod domain with different tail domains by exon shuffling.

Membrane-enclosed crystals in Dictyostelium discoideum cells, consisting of developmentally regulated proteins with sequence similarities to known esterases.

Developing cells of Dictyostelium discoideum contain crystalline inclusion bodies. The interlattice spaces of the crystals are approximately 11 nm, and their edge dimensions vary in aggregating cells from 0.1 to 0.5 micron. The crystals are enclosed by a membrane with the characteristics of RER. To unravel the nature of the crystals we isolated them under electron microscopical control and purified the two major proteins that cofractionate with the crystals, one of an apparent molecular mass of 69 kD, the other of 56 kD. This latter protein proved to be identical with the protein encoded by the developmentally regulated D2 gene of D. discoideum, as shown by its reactivity with antibodies raised against the bacterially expressed product of a D2 fusion gene. The D2 gene is known to be strictly regulated at the transcript level and to be controlled by cAMP signals. Accordingly, very little of the 56-kD protein was detected in growth phase cells, maximal expression was observed at the aggregation stage, and the expression was stimulated by cAMP pulses. The 69-kD protein is the major constituent of the crystals and is therefore called "crystal protein." This protein is developmentally regulated and accumulates in aggregating cells similar to the D2 protein, but is not, or is only slightly regulated by cAMP pulses. mAbs specific for either the crystal protein or the D2 protein, labeled the intracellular crystals as demonstrated by the use of immunoelectron microscopy. The complete cDNA-derived amino acid sequence of the crystal protein indicates a hydrophobic leader and shows a high degree of sequence similarity with Torpedo acetylcholinesterase and rat lysophospholipase. Because the D2 protein also shows sequence similarities with various esterases, the vesicles filled with crystals of these proteins are named esterosomes.