No abstract

The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn(2+)-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn(2+) binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein.

In order to determine developmental changes in atrioventricular (PQ), ventricular depolarizing (QRS) and QT intervals of the foetal heart, we recorded foetal magnetocardiographic waveforms using a superconducting quantum interference device system in a magnetically shielded room in 150 uncomplicated foetuses of gestational age >20 wk. Recording of the QRS waveform was successful in 128 (85%) of the subjects, based on unaveraged tracings. After signal averaging of the data from these 128 cases, P waves were recognized in 102 (68%) subjects and T waves in 64 (43%). The QRS interval, ranging from 32-74 ms, showed a positive linear correlation with the gestational age, which probably reflects an increase in the number and size of myocardial cells. The PQ interval showed low correlation with the gestational age, and was rather constant, with an average value of 100 ms. The QT interval ranged from 180-302 ms, and tended to be slightly shorter during early gestation. Although the success rate of measuring the PQ and QT intervals was unsatisfactory for this methodology to prevail in a clinical setting, these values provide the basis for in utero non-invasive investigation of foetal cardiac activity by magnetocardiography.

To determine the developmental changes in the myocardial current during fetal life, and to evaluate the clinical usefulness of magnetocardiography for prenatal diagnosis of cardiac hypertrophy or enlargement, we approximated the magnitude of the one-current dipole of the fetal heart using fetal magnetocardiography (fMCG). A total of 95 fetuses with gestational age of 20 -40 wk were included in this study. fMCG was recorded with a nine-channel superconducting quantum interference device system in a magnetically shielded room. The magnitude of the dipole (Q) was calculated using an equation based on the fMCG amplitude obtained on the maternal abdomen and the distance between the maternal surface and fetal heart measured ultrasonographically. In uncomplicated pregnancies, the Q value correlated significantly with gestational age, reflecting an increase in the amount of myocardial current, i.e. myocardial mass. Moreover, the Q values in fetuses with cardiomegaly caused by various cardiovascular abnormalities tended to be higher than the normal values. Although there are some limitations of the methodology based on the half-space model, and fetal orientation may influence the magnitude of the dipole, making it smaller, fMCG recorded with a multichannel superconducting quantum interference device system is a clinically useful tool for noninvasive, prenatal, and electrical evaluation of fetal cardiac hypertrophy. (1) in 1974, the magnetic field generated by the fetal heart has been measured noninvasively with satisfactory waveforms. MCG requires no pasting of electrodes to the fetal body surface and is completely noninvasive to both fetus and mother. MCG signals from the fetal heart are considered to be minimally affected by the electrical conduction properties of the tissue around the heart (2, 3). In fact, time intervals can be obtained with satisfactory signal-to-noise ratio even after the development of vernix caseosa in the second half of gestation. A number of studies, including ours, defined the developmental changes and normal ranges of various time intervals on the fMCG in uncomplicated pregnancies (4 -6). However, the amplitude of the fMCG waveform has not been fully investigated (7), although it is another important variable used for the diagnosis of fetal heart diseases. One reason for this rare application is that the amplitude of fMCG measured on the maternal abdomen does not necessarily reflect the maximum value of myocardial current because of the effects of the depth and orientation of the heart. Furthermore, these biases are not easily corrected as the fetus may move during measurement.We attempted to approximate the magnitude of the onecurrent dipole of the fetal heart based on the maximum value of fMCG data obtained with a multichannel SQUID system and the depth of the fetal heart determined by echocardiography in normal pregnancies. We then used these control values to assess magnitude abnormalities in fetuses with cardiomegaly resulting from excessive volume loading in cardiac ventricles. Using bo...

COVID-19, caused by SARS-CoV-2, has spread worldwide with a dire disaster situation.To urgently investigate the pathogenicity of COVID-19 and develop vaccines and therapeutics, animal models that are highly susceptible to SARS-CoV-2 infection are needed. In the present study, we established an animal model highly susceptible to SARS-CoV-2 via the intratracheal tract infection in CAG-promoter-driven human angiotensin-converting enzyme 2 transgenic (CAG-hACE2) mice. The CAG-hACE2 mice showed several severe symptoms of SARS-CoV-2 infection, with definitive weight loss and subsequent death. Acute lung injury with elevated cytokine and chemokine levels was observed at an early stage of infection in CAG-hACE2 mice infected with SARS-CoV-2. The analysis of the hACE2 gene in CAG-hACE2 mice revealed that more than 15 copies of hACE2 genes were tandemly integrated into the mouse genome, supporting the high susceptibility to SARS-CoV-2. In the developed model, immunization with viral antigen or injection of plasma from immunized mice prevented body weight loss and lethality due to infection with SARS-CoV-2. These results indicate that a highly susceptible model of SARS-CoV-2 infection in CAG-hACE2 mice via the intratracheal tract is suitable for evaluating vaccines and therapeutic medicines.