Neural network analysis of contrast-enhanced ultrasonography - obtained TICs seems a promising field of development for future techniques, providing fast and reliable diagnostic aid for the clinician.

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K m for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Grampositive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T % R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.Bacterial UMP kinases represent a particular subfamily of NMP 2 kinases (1, 2). They do not share any significant sequence homology with other known NMP kinases and exist in solution as stable hexamers. A first structural model of Escherichia coli UMP kinase (3) based on the conservation of the carbamate kinase and N-acetylglutamate kinase folds (4, 5) helped to better rationalize previous site-directed mutagenesis experiments (6). The crystal structure of E. coli UMP kinase (7) indicated a similar fold between its monomers and N-acetylglutamate kinase, a dimeric enzyme (4, 5). However, the quaternary structure assembly of these two proteins is completely different (7). Deposited crystal structures of UMP kinases from other bacteria such as Pyrococcus furiosus (8), Neisseria meningitidis (Protein Data Bank code 1YBD), Hemophilus influenzae (code 2AIF), and Streptococcus pyogenes (code 1Z9D) show threedimensional structures very similar to that of the E. coli enzyme. The residues essential for binding nucleotide substrates and catalysis are conserved among all bacterial UMP kinases ( Fig. 1) (3, 9). Consequently, the active sites of these enzymes and the phosphoryl transfer mechanisms are most probably similar.Comparison of the biochemical properties of recombinant UMP kinases from Gram-negative E. coli (1, 2) and Gram-positive Streptococcus pneumoniae (10) indicated significant differences in their kinetic properties particularly in their regulation by nucleotides. Unlike the E. coli enzyme, U...

The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly. Binding of Ant-dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.

Many research teams all over the world focus their research on the SARS-CoV-2, the new coronavirus that causes the so-called COVID-19 disease. Most of the studies identify the main protease or 3C-like protease (M pro /3CL pro ) as a valid target for large-spectrum inhibitors. Also, the interaction of the human receptor angiotensin-converting enzyme 2 (ACE2) with the viral surface glycoprotein (S) is studied in depth. Structural studies tried to identify the residues responsible for enhancement/weaken virus-ACE2 interactions or the cross-reactivity of the neutralizing antibodies. Although the understanding of the immune system and the hyper-inflammatory process in COVID-19 are crucial for managing the immediate and the long-term consequences of the disease, not many X-ray/NMR/cryo-EM crystals are available. In addition to 3CL pro , the crystal structures of other nonstructural proteins offer valuable information for elucidating some aspects of the SARS-CoV-2 infection. Thus, the structural analysis of the SARS-CoV-2 is currently mainly focused on three directions—finding M pro /3CL pro inhibitors, the virus-host cell invasion, and the virus-neutralizing antibody interaction. Electronic supplementary material The online version of this article (10.1007/s10930-020-09933-w) contains supplementary material, which is available to authorized users.