Construction of a family of artificial genes of human insulin

Using oligonucleotide-directed mutagenesis and chemical-enzymatic DNA synthesis, genes for A and B insulin chains, C-peptide and Tyr-C-peptide have been constructed starting from synthetic gene for human proinsulin synthesized earlier. The genes for human preproinsulin, mini-proinsulin, single-chain insulin and their modifications were also synthesized. The constructions obtained were cloned in plasmid vectors.     

A new approach to the study of genetic variability of complex characters

A new approach to multivariate genetic analysis of complex organismal traits is developed. It is based on examination of the distribution of parental strains and the F1 and F2 hybrids in a multidimensional space, and the determination of the directions corresponding to heterozygosity, epistatic and additive gene effects. The effect of heterozygosity includes variability produced by interaction between and within heterozygous loci. The additive gene effects and the remaining epistatic interactions between the homozygous loci can be visualized separately from the effects of heterozygosity by an appropriate projection of the points in multidimensional space. In all, 20 morphological, physiological and behavioural characters and 21 craniometric measures were studied in crosses between two laboratory rat strains. Linear combinations of craniometric and of morphophysiological characters with a high narrow-sense heritability could be identified. These combinations characterized the organismal stress response, which had been selected for in one of the strains. The prospects for the practical application of the new approach and also for the evaluation of the contribution of the genetic diversity to phenotypic variability in animals in natural populations are discussed.     

Virtual electrode polarization in the far field: implications for external defibrillation

We recently suggested that failure of implantable defibrillation therapy may be explained by the virtual electrode-induced phase singularity mechanism. The goal of this study was to identify possible mechanisms of vulnerability and defibrillation by externally applied shocks in vitro. We used bidomain simulations of realistic rabbit heart fibrous geometry to predict the passive polarization throughout the heart induced by external shocks. We also used optical mapping to assess anterior epicardium electrical activity during shocks in Langendorff-perfused rabbit hearts (n = 7). Monophasic shocks of either polarity (10-260 V, 8 ms, 150 microF) were applied during the T wave from a pair of mesh electrodes. Postshock epicardial virtual electrode polarization was observed after all 162 applied shocks, with positive polarization facing the cathode and negative polarization facing the anode, as predicted by the bidomain simulations. During arrhythmogenesis, a new wave front was induced at the boundary between the two regions near the apex but not at the base. It spread across the negatively polarized area toward the base of the heart and reentered on the other side while simultaneously spreading into the depth of the wall. Thus a scroll wave with a ribbon-shaped filament was formed during external shock-induced arrhythmia. Fluorescent imaging and passive bidomain simulations demonstrated that virtual electrode polarization-induced scroll waves underlie mechanisms of shock-induced vulnerability and failure of external defibrillation.     

Mechanisms of enhanced shock-induced arrhythmogenesis in the rabbit heart with healed myocardial infarction

Shock-induced vulnerability and defibrillation have been mostly studied in structurally normal hearts. However, defibrillation therapy is normally applied to patients with diseased hearts, frequently those with prior myocardial infarction (MI). Shock-induced vulnerability and defibrillation have not been well studied under this condition. We sought to examine the mechanisms of shock-induced arrhythmogenesis and arrhythmia maintenance in a rabbit model of healed MI (4 wk or more postinfarction). Ligation of the lateral division or posterolateral division of the left coronary artery at a level of 40-70% from the apex was performed 53 +/- 21 days before acute experiments. Shock-induced vulnerability was assessed in infarcted (n = 8) and structurally normal (n = 8) hearts by delivering internal monophasic shocks at different shock strengths and delivery phases. Electrical activities from the anterior epicardium during shock application and during shock-induced arrhythmias were optically recorded and quantitatively analyzed. Ligation resulted in a transmural left ventricular free wall infarction mainly located at the apical region with a consistent endocardial border zone (BZ) as confirmed by histological studies. There were significant increases in the incidence, severity, and duration of shock-induced arrhythmias in the infarcted hearts versus controls due to 1) postshock break-excitation wavefronts that frequently originated near the infarction BZ and 2) the existence of an infarction BZ that created an anatomic reentry pathway and facilitated arrhythmia maintenance. In conclusion, the infarction BZ contributes to both increased shock-induced arrhythmogenesis and arrhythmia maintenance in the rabbit model of healed MI.     

The Aspergillus nidulans sldI(RAD50) gene interacts with bimE(APC1), a homologue of an anaphase-promoting complex subunit

The Mre11-Rad50-Nbs1 protein complex has emerged as a central component in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA damage detection to DNA repair and cell cycle checkpoint functions. We have identified Aspergillus nidulans sldI1444D mutant in a screen for dynein synthetic lethals. The sldI(RAD50) gene was cloned by complementation of the sporulation deficiency phenotype of this mutant. A transversion G-->C at the position 2509 (Ala-692-Pro amino acid change) in the sldI1444D mutant causes sensitivity to several DNA-damaging agents. The mutation sldI1 occurs at the CXXC hinge domain of Rad50. We have deleted part of the coiled-coil and few amino acids of the Rad50-Mre11 interaction region and assessed several phenotypic traits in this deletion strain. Besides sensitivity to a number of DNA-damaging agents, this deletion strain is also impaired in the DNA replication checkpoint response, and in ascospore viability. There is no delay of the S-phase when germlings of both sldI (RAD50) and mreA(MRE11) inactivation strains were exposed to the DNA damage caused by bleomycin. Transformation experiments and Southern blot analysis indicate homologous recombination is dependent on scaA(NBS1) function in the Mre11 complex. There are epistatic and synergistic interactions between sldI( RAD50) and bimE(APC1) at S-phase checkpoints and response to hydroxyurea and UV light. Our results suggest a possible novel feature of the Mre11 complex in A. nidulans, i.e. a relationship with bimE (APC1).     

Two mechanisms of protein folding: A theoretical analysis

In the current paper, two presumable mechanisms of protein folding are discussed; one of them, the “nucleation mechanism”, is exemplified by 3β-corner domains containing proteins, while the other, “structural units mechanism”, is exemplified by serine proteases. The analysis of the spatial structure of 3β-corners and of the common features of the amino acid sequences encoding them made it possible to conclude, that 3β-corners are capable of assuming their unique structures on their own and can serve as the nuclei or preformed structural units in the process of protein folding. The high order protein structures may be obtained by the further successive addition of β strands to a 3β-corner acting as a nucleus, according to certain rules and restrictions. On the other hand, 3β-corner may serve as a preformed structural unit, and the association of two 3β-corners may result in the formation of such 3D structures, which can be found in the domains of serine proteases and similar proteins.     

Regeneration of mammalial plantar skin]

Two sole tubers of rat's hind leg were excised together with the surrounding skin and one of the hind tubers of hind and fore legs in hedgehogs were excised without the surrounding skin. During healing the skin wounds in rats and hedgehogs new skin proved to form with the papillary layer typical of the sole skin. In hedgehogs the new skin covered the regenerated sole tuber; in rats the sole tubers failed to regenerate.