A bioluminescent method of determining antibiotic sensitivity of microbial cells in septic blood]

Previous studies have shown that adhesion molecules play a crucial role in leukocyte-endothelium interactions that occur during myocardial ischemia and reperfusion. We assessed the plasma levels of the soluble form of E-selectin (sE-selectin) and intercellular adhesion molecule-1 (sICAM-1) in 15 patients with acute myocardial infarction (AMI) and in 15 controls with chronic stable angina. In patients with AMI, the levels of sE-selectin and sICAM-1 increased significantly during the first 8 h after infarction and subsequently decreased. Soluble E-selectin levels were inversely related to the peak plasma levels of creatine kinase-MB (CK-MB), and the time course of their appearance in plasma correlated with that of neutrophil count and plasma D-dimer. In individual patients, peak and mean sICAM-1 levels correlated respectively with plasma D-dimer concentrations and monocyte count, but no correlation were found when their time courses were analyzed. Eight hours after symptom onset, the mean plasma sE-selectin levels were higher in patients with AMI than in those with stable angina, whereas no significant differences were found in mean plasma sICAM-1 levels between the two groups at every time analyzed. In the acute phase of MI (a) sE-selectin and sICAM-1 levels increase during the first 8 h and subsequently decrease; (b) the increase in sE-selectin probably reflects activation of endothelial cells, correlates with other inflammatory and coagulation parameters, and is inversely related to the degree of myocardial damage; and (c) sICAM-1 plasma levels do not represent a good marker of "cell activation" because they reflect activation of different cells and may be affected by different conditions.     

RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations

Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate. The extracellular ribonuclease from Bacillus intermedius (binase, RNase Bi) shares a common mechanism for RNA hydrolysis with mammalian RNases. Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage. Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GpC and guanosine 2',3'- cyclic phosphate (cGMP) substrates were studied. The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.      

Optimization of Computations in the System of Computer Simulation fSim

A computational model of the simulation system fSim is constructed in terms of graph theory. The problem of transition from multi-level models to one-level ones is considered. The principles of construction of simulation models supported by fSim under conditions of distributed computations are described. Based on the droop vector method and heuristic methods, algorithms of optimization in distributed computing systems are considered.     

beta-Fructosidases: a new superfamily of glycosyl-hydrolases

Dihydropyridines (DHPs) block L-type Ca2+ channels more potently at depolarized membrane potentials, consistent with high affinity binding to the inactivated state. Nisoldipine (a DHP antagonist) blocks the smooth muscle channel more potently than the cardiac one, a phenomenon observed not only in native channels but also in expressed channels. We examined whether this tissue specificity was attributable to differences of inactivation in the two channel types. We expressed cardiac or smooth muscle alpha1C subunits in combination with beta2a and alpha2/delta subunits in human embryonic kidney cells, and used 2 mM Ca2+ as the permeant ion. This system thus reproduces the in vivo topology and charge carrier of the channels while facilitating comparison of the two alpha1C splice variants. Both voltage-dependent and isoform-specific sensitivity of 10 nM nisoldipine inhibition of the channel were demonstrated, with the use of -100 mV as the holding potential for fully reprimed channels and -65 mV to populate the inactivated state. Under drug-free conditions, we characterized fast inactivation (1-sec prepulses) and slow inactivation (3 min prepulses) in the two isoforms. Inactivation parameters were not statistically different in the two channel isoforms; if anything, cardiac channels tended to inactivate more than the smooth muscle channels at relevant voltages. Likewise, the voltage-dependent activation was identical in the two isoforms. We thus conclude that the more potent nisoldipine inhibition of smooth muscle versus cardiac L-type Ca2+ channels is not attributable to differences in channel inactivation or activation. Intrinsic, gating-independent DHP receptor binding affinity differences must be invoked to explain the isoform-specific sensitivity of the DHP block.     

Characteristics of microwave and thermal heating of samples of cell suspensions and solutions of several compounds

The influence of microwaves and heat on dynamics of heating of samples of intact and inactivated bacteria Escherichia coli and solutions of some basic molecular components of cell was investigated. It was shown that microwaves induce different dynamics of heating of all samples. On the contrary, thermal action induces identical dynamics of heating of all this samples except vegetable oil which was heated more intensively.     

Model building of a thermolysin-like protease by mutagenesis

The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub). An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus thermoproteolyticus (thermolysin) and Bacillus cereus as templates. The largest portion of NP-sub could be modelled satisfactorily, using standard techniques, but several surface-located regions could only be modelled with a high degree of uncertainty. In order to make the model more reliable in these regions a 'model building by mutagenesis' approach was adopted. Mutations were designed such that their effect on thermal stability could indicate how their local environment should be modelled. This approach provided insight in the local structure of several regions in NP-sub that were hard to model on the basis of homology with the two known structures alone.