Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase.

A genetic block was introduced in the first condensation step of the polyketide biosynthetic pathway that leads to the formation of 6-deoxyerythronolide B (6-dEB), the macrocyclic precursor of erythromycin. Exogenous addition of designed synthetic molecules to small-scale cultures of this null mutant resulted in highly selective multimilligram production of unnatural polyketides, including aromatic and ring-expanded variants of 6-dEB. Unexpected incorporation patterns were observed, illustrating the catalytic versatility of modular polyketide synthases. Further processing of some of these scaffolds by postpolyketide enzymes of the erythromycin pathway resulted in the generation of novel antibacterials with in vitro potency comparable to that of their natural counterparts.

An efficient antibody-catalyzed aminoacylation reaction.

An antibody generated against a neutral phosphonate diester transition-state analog was found to catalyze the aminoacylation of the 3'-hydroxyl group of thymidine with an alanyl ester. A comparison of the apparent second-order rate constant of the antibody-catalyzed reaction [5.4 x 10(4) molar-1 minute-1 (M-1 min-1)] with that of the uncatalyzed reaction (2.6 x 10(-4) M-1 min-1) revealed this to be a remarkably efficient catalyst. Moreover, although the concentration of water (55 M) greatly exceeds that of the secondary alcohol, the antibody selectively catalyzes acyl transfer to thymidine. The antibody exhibits sequential binding, with Michaelis constants of 770 microM and 260 microM for acyl acceptor and donor, respectively, and a dissociation constant of 240 pM for hapten. This antibody-catalyzed reaction provides increased insight into the requirements for efficient aminoacylation catalysts and may represent a first step toward the generation of "aminoacyl transfer RNA synthetases" with novel specificities.

High frequency of submicroscopic genomic aberrations detected by tiling path array comparative genome hybridisation in patients with isolated congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect and affects nearly 1% of newborns. The aetiology of CHD is largely unknown and only a small percentage can be assigned to environmental risk factors such as maternal diseases or exposure to mutagenic agents during pregnancy. Chromosomal imbalances have been identified in many forms of syndromic CHD, but very little is known about the impact of DNA copy number changes in non-syndromic CHD. METHOD: A sub-megabase resolution array comparative genome hybridisation (CGH) screen was carried out on 105 patients with CHD as the sole abnormality at the time of diagnosis. RESULTS: There were 18 chromosomal changes detected, which do not coincide with common DNA copy number variants, including one de novo deletion, two de novo duplications and eight familial copy number variations (one deletion and seven duplications). CONCLUSIONS: Our data show that submicroscopic deletions and duplications play an important role in the aetiology of this condition, either as direct causes or as genetic risk factors for CHD. These findings have immediate consequences for genetic counselling and should pave the way for the elucidation of the pathogenetic mechanisms underlying CHD.

The scope of antibody catalysis.

New strategies for hapten design have led to antibodies that catalyze reactions by increasingly complex mechanisms and with large increases in catalytic rate. Rational design has also been used to elicit catalytic antibodies for difficult chemical transformations as well as reactions for which no enzyme is known. These experiments have demonstrated the chemical potential of large combinatorial libraries that have been given appropriate mechanistic instruction.

Relationship between cumulative anthracycline dose and late cardiotoxicity in childhood acute lymphoblastic leukemia.

PURPOSE: Late anthrocycline cardiotoxicity after treatment for childhood cancer is common and often progressive. A safe anthracycline dose that will not result in late cardiac abnormalities has not been established due to the limited dose ranges used in existing studies. PATIENTS AND METHODS: To determine the relationship between cumulative anthracycline dose and late cardiotoxicity, we performed echocardiograms on 189 survivors of childhood acute lymphoblastic leukemia a median of 8.1 years (range, 2.0 to 23.4) after completion of anthracycline therapy. Patients were treated according to protocols that used widely varying cumulative anthracycline doses, but comparable nonanthracycline chemotherapy. Patients were divided into four groups based on the city of treatment and cumulative anthracycline dose: Copenhagen, 0 to 23 mg/m2 (n = 32); Boston, 45 mg/m2 (n = 17); Copenhagen, 73 to 301 mg/m2 (n = 53); and Boston, 244 to 550 mg/m2 (n = 87). Left ventricular dimension and fractional shortening were adjusted for sex and age or body-surface area through use of a control population (n = 296), and then compared among the four groups. RESULTS: Mean left ventricular dimension was significantly increased in the high-dose Boston group (observed:predicted value, 4.57 cm:4.45 cm; P = .002) and significantly higher than in the two Copenhagen groups. In the three lower-dose groups, there was no significant increase in mean left ventricular dimension, and the groups were not significantly different from each other. Similarly, the mean left ventricular fractional shortening was significantly depressed in the high-dose Boston group (observed:predicted value, 29.0%:33.8%; P = .0001) and significantly lower than in the three lower-dose groups. CONCLUSION: Depressed left ventricular fractional shortening and left ventricular dilatation were uncommon years after treatment of childhood leukemia when cumulative anthracycline doses were < or = 300 mg/m2.

New directions in metabolic engineering.

Metabolic engineering is a rapidly evolving field. The term typically refers to the genetic modification of cellular biochemistry to introduce new properties or modify existing ones. Recent progress in genetics, molecular biology, microbiology and chemistry are driving advances in this field. Many well-studied areas continue to yield exciting results and new problems and technologies are constantly being developed.

A functional chimeric modular polyketide synthase generated via domain replacement.

BACKGROUND: Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules. RESULTS: To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated. CONCLUSIONS: These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.

Molecular recognition of diketide substrates by a beta-ketoacyl-acyl carrier protein synthase domain within a bimodular polyketide synthase.

BACKGROUND: Modular polyketide synthases (PKSs) are large multifunctional proteins that catalyze the biosynthesis of structurally complex bioactive products. The modular organization of PKSs has allowed the application of a combinatorial approach to the synthesis of novel polyketides via the manipulation of these biocatalysts at the genetic level. The inherent specificity of PKSs for their natural substrates, however, may place limits on the spectrum of molecular diversity that can be achieved in polyketide products. With the aim of further understanding PKS specificity, as a route to exploiting PKSs in combinatorial synthesis, we chose to examine the substrate specificity of a single intact domain within a bimodular PKS to investigate its capacity to utilize unnatural substrates. RESULTS: We used a blocked mutant of a bimodular PKS in which formation of the triketide product could occur only via uptake and processing of a synthetic diketide intermediate. By introducing systematic changes in the native diketide structure, by means of the synthesis of unnatural diketide analogs, we have shown that the ketosynthase domain of module 2 (KS2 domain) in 6-deoxyerythronolide B synthase (DEBS) tolerates a broad range of variations in substrate structure, but it strongly discriminates against some others. CONCLUSIONS: Defining the boundaries of substrate recognition within PKS domains is crucial to the rationally engineered biosynthesis of novel polyketide products, many of which could be prepared only with great difficulty, if at all, by direct chemical synthesis or semi-synthesis. Our results suggest that the KS2 domain of DEBS1 has a relatively relaxed specificity that can be exploited for the design and synthesis of medicinally important polyketide products.

One-stage repair of truncus arteriosus, CAVC, and TAPVC.

An infant with truncus arteriosus, complete atrioventricular canal, and total anomalous pulmonary venous connection successfully underwent one-stage complete repair. Residual mitral valve regurgitation required reoperation after 12 days. The patient is doing well at 6 months' follow-up. Echocardiography demonstrates no residual defects, competent atrioventricular valves, and normal pulmonary pressure. This case illustrates the potential for successful one-stage repair even of associated complex heart defects involving venous, intracardiac, and arterial pathways.

A single strand conformation polymorphism/heteroduplex (SSCP/HD) method for detection of mutations in 15 exons of the KVLQT1 gene, associated with long QT syndrome.

Congenital long QT syndrome (LQTS) is characterised by prolongation of the QT interval on ECG and cardiac arrhythmias, syncopes and sudden death. A rapid and reliable genetic diagnosis of the disease may be of great importance for diagnosis and treatment of LQTS. Mutations in the KVLQT1 gene, encoding a potassium-channel subunit of importance for the depolarisation of cardiac myocytes, is believed to be associated with 50% of all LQTS cases. Our data confirms that KvLQT1 isoform 1 is encoded by 16 exons, and not 15, as reported previously. We have used genomic DNA sequences to design intronic PCR primers for amplification of 15 exons of KVLQT1 and optimised a non-radioactive single stranded conformation polymorphism/heteroduplex (SSCP/HD) method for detection of mutations in KVLQT1. The sensitivity of the method was 100% when it was tested on 15 in vitro constructed mutants. By multiplexing the PCR amplification of KVLQT1, it is possible to cover all 15 exons in four PCR reactions.

Is the arterial switch operation still a challenge in small centers?

OBJECTIVE: In the last years, major changes as regards timing for operation, surgical technique, and perioperative care determined a great improvement in the arterial switch operation (ASO) allowing excellent mid-term results in a few leading centers. This stimulated the widespread adoption of ASO as procedure of choice for transposition of the great arteries (TGA), even in small institutions. We reviewed our early experience with ASO in an attempt to evaluate its safety in a small center. METHODS: Since April 1992, 39 consecutive patients underwent TGA repair by ASO in our department. There were 27 patients with simple TGA, 8 with TGA and VSD and 4 with Taussig-Bing heart and aortic coarctation. Median age and weight at operation were 7 days and 3.5 kg, respectively. Neonatal repair was performed in 34 patients. In accordance with the Planché coronary classification, type I was encountered in 21 patients, type II in 4 and type III in 14. Several modifications of the original technique were used, mainly regarding coronary relocation, pulmonary artery reconstruction and approaches for associated VSD closure and aortic arch repair. RESULTS: Early mortality was 2.6% (n = 1), the only operative death being related to unsatisfactory coronary relocation. Since modified ultrafiltration was adopted, mean ICU stay decreased from 5 +/- 4 days (n = 21) to 2 +/- 1 days (n = 17) (P < 0.05). Three patients required reoperation for residual ASD and/or VSD closure. There were no late deaths. After a mean follow-up of 26 +/- 15 months all survivors are thriving and are currently asymptomatic. CONCLUSIONS: Although this series is rather small, most of the major coronary anomalies and complex anatomic associations were encountered. This experience suggests that neonatal repair of TGA by ASO can be safely accomplished even in small centers. Modified ultrafiltration appears to improve the early outcome of neonates undergoing ASO.

[Prenatal detection of congenital heart disease]. / Praenatal opsporing af kongenit hjertesygdom.

Foetal echocardiography is a sensitive and specific method of investigation for prenatal detection of cardiac malformations. Experience has shown that brief echocardiographic screening can demonstrate, directly or indirectly, the presence of or probability of serious cardiac disease in the foetus as early as the 18th-20th weeks of pregnancy. The method of examination and its limitations are mentioned and it is recommended that screening should be carried out as part of other forms of obstetric ultrasonic scanning. Early prenatal diagnosis of severe structural cardiac malformations provide the possibility for selective termination of pregnancy, optimal pre- and perinatal observation and treatment. The possibility of prenatal diagnosis of serious cardiac malformations increases the value of obstetric ultrasonic scanning and may be included among the advantages of offering this form of investigation to all pregnant women.

[Pulmonary hypertension in children treated with ventriculo-atrial shunt in hydrocephalus]. / Pulmonal hypertension hos børn behandlet med ventrikulo-atrial shunt for hydrocephalus.

Four infants treated with ventriculo-atrial shunt for hydrocephalus developed sudden pulmonary hypertension, several years after the first operation. All infants died in spite of replacement of the distal shunt and AK treatment. At necropsy, old microemboli were found in the small lung vessels in two infants whereas the two other infants were suspected of having the same lesions from clinical investigations. The overall frequency for this complication was estimated to be 6.7% (95% CI 1.4-14.3%).

[Kawasaki disease. Incidence in Denmark during the period 1981-1990]. / Kawasaki's sygdom. Forekomst i Danmark i perioden 1981-1990.

The aim of the study was to estimate the incidence of Kawasaki's disease and to describe the clinical manifestations and the prognosis of the disease in children in Denmark during the period 1981-1990. In a register of all hospital admissions in Denmark, 99 patients were registered as having Kawasaki's disease. Of those, 89 patients fulfilled the clinical diagnostic criteria. The mean incidence of the ten year period was 1.0 per 100,000 children per year. The incidence of the disease decreased in the age group from zero to seven years after which it was rarely observed. Typical laboratory findings were hypersedimentation, leucocytosis and thrombocytosis. Eight children had cardiomegaly and three had electrocardiographic changes. Echocardiography was performed in 66 patients showing coronary artery aneurysms in nine patients (14%) (six boys and three girls). No further cardiac complications were found.

[Percutaneous pulmonary balloon valvuloplasty in Denmark]. / Perkutan pulmonal ballonvalvuloplastik i Danmark.

Percutaneous balloon valvuloplasty of valvular pulmonary stenosis (PPB) was carried out for the first time in 1982 and is now regarded as the primary method of treatment of this condition. The results of the first PPB treatments in Denmark are presented here. PPB was planned in 28 patients and was carried out in 25 (22 children and 3 adults). PPB was carried out on two occasions in one patient. Twenty-three patients had isolated valvular pulmonary stenosis and two patients had Fallot's anomaly. No complications of significance occurred after the treatments. The average gradient for all dilatations was 77 +/- 24 mm Hg prior to and 36 +/- 23 (p less than 0.0001) immediately after PPB. The gradient was reduced by more than 50% in 68% of the patients. In 14 patients, the gradients over the pulmonary valve was measured by Doppler technique or by cardiac catheterization greater than 6 months after PPB. In these patients, the average gradient was 69 +/- 21 mm Hg prior to PPB, 29 +/- 12 mm Hg (p less than 0.0001) immediately after PPB and 27 +/- 9 mm Hg (p less than 0.0001) at the most recent control examination, on an average 12 months (range 6-24 months) after PPB. In the same patient group, significant reduction of the electrocardiographic right-sided hypertrophy was found at the most recent control examination. It is concluded that PPB is an effective and safe treatment of valvular pulmonary stenosis.

[In vitro fertilization and embryo transfer. 2. The first 44 deliveries of 55 infants]. / In vitro-fertilisation og embryotransplantation. 2. De første 44 fødsler omfattende 55 børn.

During the period 1.9.1985-21.2.1988, 143 patients commenced and completed treatment with in vitro fertilization and embryo-transfer (IVF-ET) in Rigshospitalet, Copenhagen. Forty-four out of the 143 patients were delivered of a total of 55 infants. There were 35 single deliveries, seven twin deliveries and two triplet deliveries. Seven women were admitted with threatened abortion and three women developed preeclampsia, one of these was severe. On an average, the deliveries occurred in the 38th week of pregnancy and the average weight of the singletons was 3,020 g. Ten women were delivered preterm and seven of the singletons had birth weights under 2,500 g. The frequency of caesarean section was 34% for singleton deliveries and 43% for all of the deliveries. Thirty-one boys and 24 girls were delivered, all of whom had normal karyotypes. One infant had a cleft lip but no other malformations. Three premature infants required brief respirator treatment. All of the infants were thriving on discharge. This material is still too limited to permit drawing of definite conclusions but it suggests, however, just as in other IVT-ET investigations, that the infants are born slightly earlier and weigh slightly less than average neonates, but that they are otherwise normal. In order to illustrate these problems further, the authors have commenced a more extensive prospective investigation with matched control persons.