Importance of extracutaneous organ involvement in determining the clinical severity and prognosis of incontinentia pigmenti caused by mutations in the IKBKG gene.

Incontinentia pigmenti (IP) is a rare X-linked skin disease caused by mutations in the IKBKG gene, which is required for activation of the nuclear factor-kappa B signalling pathway. Multiple systems can be affected with highly variable phenotypic expressivity. We aimed to clarify the clinical characteristics observed in molecularly confirmed Korean IP patients. The medical records of 25 females confirmed as IP by molecular genetic analysis were retrospectively reviewed. The phenotypic score of extracutaneous manifestations was calculated to assess the disease severity. The IKBKG gene partial deletion or intragenic mutations were investigated using long-range PCR, multiplex ligation-dependent probe amplification and direct sequencing methods. Among the 25 individuals, 18 (72%) were sporadic cases. All patients showed typical skin manifestations at birth or during the neonatal period. Extracutaneous findings were noted in 17 (68%) patients; ocular manifestations (28%), neurological abnormalities (28%), hair abnormalities (20%), dental anomalies (12%), nail dystrophy (8%). The common exon 4-10 IKBKG deletion was observed in 20 (80%) patients. In addition, five intragenic sequence variants were identified, including three novel variants. The phenotype scores were highly variable, ranging from abnormal skin pigmentation only to one or more extracutaneous features, although no significant difference was observed for each clinical characteristic between the group with sequence variants and that with common large deletion. Our cohort with IP showed heterogeneity of extracutaneous manifestations and high incidence of sporadic cases. Long-term monitoring with multidisciplinary management is essential for evaluating the clinical status, providing adequate genetic counselling and understanding the genotype-phenotype correlation in IP.

Essential Role of the Linker Region in the Higher Catalytic Efficiency of a Bifunctional MsrA-MsrB Fusion Protein.

Many bacteria, particularly pathogens, possess methionine sulfoxide reductase A (MsrA) and B (MsrB) as a fusion form (MsrAB). However, it is not clear why they possess a fusion MsrAB form rather than the separate enzymes that exist in most organisms. In this study, we performed biochemical and kinetic analyses of MsrAB from Treponema denticola (TdMsrAB), single-domain forms (TdMsrA and TdMsrB), and catalytic Cys mutants (TdMsrAB(C11S) and TdMsrAB(C285S)). We found that the catalytic efficiency of both MsrA and MsrB increased after fusion of the domains and that the linker region (iloop) that connects TdMsrA and TdMsrB is required for the higher catalytic efficiency of TdMsrAB. We also determined the crystal structure of TdMsrAB at 2.3 Å, showing that the iloop mainly interacts with TdMsrB via hydrogen bonds. Further kinetic analysis using the iloop mutants revealed that the iloop-TdMsrB interactions are critical to MsrB and MsrA activities. We also report the structure in which an oxidized form of dithiothreitol, an in vitro reductant for MsrA and MsrB, is present in the active site of TdMsrA. Collectively, the results of this study reveal an essential role of the iloop in maintaining the higher catalytic efficiency of the MsrAB fusion enzyme and provide a better understanding of why the MsrAB enzyme exists as a fused form.

Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine.

Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. (75)Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher Km than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec.

Structural Insights into a Bifunctional Peptide Methionine Sulfoxide Reductase MsrA/B Fusion Protein from Helicobacter pylori.

Methionine sulfoxide reductase (Msr) is a family of enzymes that reduces oxidized methionine and plays an important role in the survival of bacteria under oxidative stress conditions. MsrA and MsrB exist in a fusion protein form (MsrAB) in some pathogenic bacteria, such as Helicobacter pylori (Hp), Streptococcus pneumoniae, and Treponema denticola. To understand the fused form instead of the separated enzyme at the molecular level, we determined the crystal structure of HpMsrABC44S/C318S at 2.2 Å, which showed that a linker region (Hpiloop, 193-205) between two domains interacted with each HpMsrA or HpMsrB domain via three salt bridges (E193-K107, D197-R103, and K200-D339). Two acetate molecules in the active site pocket showed an sp2 planar electron density map in the crystal structure, which interacted with the conserved residues in fusion MsrABs from the pathogen. Biochemical and kinetic analyses revealed that Hpiloop is required to increase the catalytic efficiency of HpMsrAB. Two salt bridge mutants (D193A and E199A) were located at the entrance or tailgate of Hpiloop. Therefore, the linker region of the MsrAB fusion enzyme plays a key role in the structural stability and catalytic efficiency and provides a better understanding of why MsrAB exists in a fused form.

Oral contrast for abdominal computed tomography in children: the effects on gastric fluid volume.

BACKGROUND: Oral enteric contrast medium (ECM) is frequently administered to achieve visualization of the gastrointestinal tract during abdominal evaluation with computed tomography (CT). Administering oral ECM less than 2 hours before sedation/anesthesia violates the nothing-by-mouth guidelines and in theory may increase the risk of aspiration pneumonia. In this study we measured the residual gastric fluid when using a protocol in which ECM is administered up to 1 hour before anesthesia/sedation. We hypothesized that patients receiving ECM 1 hour before anesthesia/sedation would have residual gastric fluid volume (GFV) >0.4 mL/kg. METHODS: Anesthesia and radiology reports, CT images, and department incident reports were reviewed between January 2005 and June 2009 for all patients who required sedation/anesthesia for abdominal CT. For each patient, the volume of contrast or stomach fluid was calculated using a region of interest outlining the stomach portion containing high-attenuation fluid and low-attenuation of other gastric contents. Information obtained from anesthesia/sedation reports included demographic characteristics, presenting pathology, drugs used for anesthesia/sedation induction and maintenance, airway interventions, method for securing endotracheal tube, and complications related to ECM administration, including oxygen desaturation, vomiting, coughing, bronchospasm, laryngospasm, and aspiration. RESULTS: We identified 365 patients (mean age = 32 months; range = 0.66 to 211.10 months) who received oral/IV contrast material before anesthesia/sedation for abdominal CT and 47 patients (mean age = 52 months; range = 0.63 to 215.84 months) who received only IV contrast material and followed the traditional fast. For those who received oral contrast, the mean contrast volume administered was 18.10 mL/kg (range = 1.5 to 82.76 mL/kg). The median GVF 1 hour after completing the oral contrast was significantly higher than that in patients who received only IV contrast (0.38 mL/kg vs. 0.15 mL/kg, P = 0.0049). GFV exceeded 0.4 mL/kg in 189 patients (178 of 365 [49%] in the oral contrast group vs. 11 of 47 [23%] in the IV contrast group) (χ(2) = 10.7874, P = 0.0010). Among those who received oral contrast, 207 patients had general anesthesia and 158 patients had deep sedation. Two cases of vomiting were reported in the general anesthesia group with no evidence of pulmonary aspiration identified. CONCLUSION: For children receiving an abdominal CT, the residual GFV exceeded 0.4 mL/kg in 49% (178/365) of those who received oral ECM up to 1 hour before anesthesia/sedation in comparison with 23% (11/47) of those who received IV-only contrast.