Testicular Torsion in Children. Diagnostic and Prognostic Potential of Ultrasonography

Introduction: Testicular torsion is the most urgent case in pediatric andrology that requires emergency care due to the high sensitivity of testicular tissue to hypoxia.Objective: To determine statistically significant parameters indicating diagnostic significance of some ultrasonographic testicular torsion symptoms in children.Materials and methods: Ultrasonography was performed in 202 children with testicular torsion (main group) and 449 patients (control group) aged from 2 months to 17 years 11 months and 28 days. During statistical processing we determined the mean values, their standard deviation, sampling errors, significance of differences in the mean values, correlation coefficient and did ROC analysis. For an objective ultrasonographic assessment of testicular shape, we implemented the coefficient of rounding – ko = (testicular length – testicular thickness) / testicular length – and the parameter Δko calculated as the difference between ko for the affected side and the contralateral one.Results and discussion: Testicular torsion was more common in children above 12 years of age (84.7%). The ko was 0.160 ± 0.007 in the group of children with testicular torsion and 0.037±0.003 in the control group (t = 16.92, p >> .001). The ROC analysis proved the Δko to be highly informative: AUC was 0.886. The Δko was 0.130 ± 0.008 for patients with favorable treatment outcomes and 0.19 ± 0.01 (p < 0.001) for those with unfavorable outcomes. In most cases (71), the whirlpool sign was visualized with preserved blood flow. In such a case, the probability of the favorable treatment outcome was 74.6%. The whirlpool sign visualization was significantly less common (31 cases) without Doppler ultrasonography; favorable treatment outcomes were achieved in 54.8%. Of 22 patients with the undetected whirlpool sign, favorable treatment outcomes were possible only in 40.9%.Conclusions: The coefficient of rounding ko can be used as an auxiliary criterion for the early diagnosis of testicular torsion. With Δko = 0.08, the sensitivity of the parameter in respect of testicular torsion is 79.2%, and specificity is 85.7%. With Δko = 0.16, the specificity of the parameter is almost 100% with a 45.0% decrease of the sensitivity. Whirlpool sign detection using color Doppler ultrasonography is a positive prognostic factor

Combined computational and biochemical study reveals the importance of electrostatic interactions between the ‘‘pH sensor’’ and the cation binding site of the sodium/proton antiporter NhaA of Escherichia coli

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. The crystal structure of NhaA has provided a basis to explore the mechanism of ion exchange and it's unique regulation by pH. Here, the mechanism of the pH activation of the antiporter is investigated through functional and computational studies of several variants with mutations in the ion-binding site (D163, D164). The most significant difference found computationally between the wild type antiporter and the active site variants, D163E and D164N, are low pKa values of Glu78 making them insensitive to pH. Although in the variant D163N the pKa of Glu78 is comparable to the physiological one, this variant cannot demonstrate the long-range electrostatic effect of Glu78 on the pH-dependent structural reorganization of trans-membrane helix X and, hence, is proposed to be inactive. In marked contrast, variant D164E remains sensitive to pH and can be activated by alkaline pH shift. Remarkably, as expected computationally and discovered here biochemically, D164E is viable and active in Na+/H+ exchange albeit with increased apparent KM. Our results unravel the unique electrostatic network of NhaA that connect the coupled clusters of the ‘‘pH sensor’’ with the binding site, which is crucial for pH activation of Nha

Transmembrane Segment II of NhaA Na⁺/H⁺ Antiporter Lines the Cation Passage, and Asp⁶⁵ Is Critical for pH Activation of the Antiporter

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 5.5– 8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent Km to Na+ and Li+ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [14C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent Km of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp65 negative charge for pH activation of the antiporter

A L-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily PRODUCTION, FUNCTIONAL CHARACTERIZATION, AND STRUCTURE MODELING

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of L-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of L-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40μM at pH 5.9 and is highly selective; no evidence was found for the binding of L-arginine, L-ornithine, L-2,4-diaminobutyric acid, and L-alanine. D-Lysine is bound 45 times more weakly than its L-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for L-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed

Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum

Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe–2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs

High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: New insights into the active site and the proton transfer pathways

The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 Å resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a3 iron and CuB of the active site is fitted best by a peroxogroup or a chloride ion. Two waters or OH− groups do not fit, one water (or OH−) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the CuB ligand His326 is present in the imidazolate for

A D-Pathway Mutation Decouples the Paracoccus denitrificans Cytochrome c Oxidase by Altering the Side-Chain Orientation of a Distant Conserved Glutamate

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa3 cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-Å resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase

Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na+/H+ antiporter NhaA

NhaA, a Na+/H+ antiporter critical for pH and Na+ homeostasis in Escherichia coli, as well as other enterobacteria and possibly Homo sapiens, was modified for fluorescence spectroscopy by constructing a functional Trp-less NhaA mutant. Purified Trp-less NhaA lacks the Trp fluorescence emission characteristic of the wild type, thereby providing a background for studying structure–function relationships in NhaA by site-directed Trp fluorescence. Two single-Trp variants in the Trp-less background (F136W and F339W) were constructed. The mutants grow on selective media, have antiport activities that are similar to Trp-less NhaA, and exhibit Trp fluorescence with three different reversible responses to Li+, Na+, and/or pH. With single Trp/F136W, a pH shift from pH 6.0 to 8.5 induces a red shift and dramatically increases fluorescence in a reversible fashion; no effect is observed when either Na+ or Li+ is added. In marked contrast, with single Trp/F339W, changes in pH do not alter fluorescence, but addition of either Na+ or Li+ drastically quenches fluorescence at alkaline pH. Therefore, a Trp at position 136 specifically monitors a pH-induced conformational change that activates NhaA, whereas a Trp at position 339 senses a ligand-induced conformational change that does not occur until NhaA is activated at alkaline pH