Congenital Colonic Atresia: Report of One Case

Colonic atresia is a very rare cause of intestinal obstruction, and surgical management is the mainstay of therapy. A case of congenital colonic atresia is reported in a full-term neonate who presented with delayed passage of meconium, abdominal distention and bilious vomiting. The present case and the pertinent literature are discussed, with an emphasis on surgical management

N-(Pyrazin-2-yl)-1,8-naphthyridin-2-amine

There are two independent molecules in the asymmetric unit of the title compound, C12H9N5, in which the C—N(amine)—C angles differ slightly [129.63 (11) and 132.02 (11)°]. In each independent molecule, an intramolecular C—H...N hydrogen bond stabilizes the molecular structure, forming an S(6) ring motif. The independent molecules are linked via an N—H...N hydrogen bond. Further N—H...N and C—H...N hydrogen bonds connect the molecules into chains along c axis. Pairs of C—H...π interactions between the chains lead to sheets parallel to the b axis. These are linked by π–π interactions between the naphthyridine and pyrazine rings [centroid–centroid separations of 3.553 (8) Å] into a three-dimensional supramolecular network

Regulation of the Abundance of Kaposi’s Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2

<div><p>The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50.</p></div

Lysine residues at positions 152 and 154 in ORF50 are critical for MDM2-mediated degradation.

<p>(A) Schematic diagram of ORF50 and ORF50 lysine mutants. The positions of 25 lysines in ORF50 are shown in the diagram (circles). Black circles represent the substitutions of lysine (K) with arginine (R) in F-ORF50. (B) Immunoblot analysis of lysine substitution mutants of ORF50. Cell lysates of 293T cells that were transfected with the indicated expression plasmids were probed with anti-FLAG antibody. (C) Effects of single- or double-lysine mutations from K124 to K243 (the middle 8 lysine clusters) on ORF50 abundance. (D) Susceptibility of ORF50 mutants to MDM2-mediated degradation. The expression plasmids encoding ORF50 mutants were cotransfected with an MDM2 expressing plasmid or control vector into 293T cells. The expression of these ORF50 mutants in the presence or absence of exogenous MDM2 was determined by immunoblotting with anti-FLAG antibody. (E) Relative protein levels of ORF50 mutants in the presence or absence of overexpressed MDM2. The bar graph summarizes densitometry data from three independent experiments. Error bars: standard deviation.</p

Both N-terminal and C-terminal regions in ORF50 are required for ubiquitination.

<p>(A) Deletion constructs of ORF50 and a summary of their intracellular localization, protein abundance and ubiquitination status. (B) Immunoblot analysis of F-ORF50, F-ORF50(1–590) and F-ORF50(357–691) in 293T cells. (C and D) Mutants F-ORF50(1–590) and F-ORF50(357–691) were evaluated for ubiquitination in 293T cells. Cells were transfected with the indicated plasmids. At 16 hr after transfection, cells were treated with MG132 for another 24 hr. Cell lysates were immunoprecipitated and immunoblotted as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005918#ppat.1005918.g006" target="_blank">Fig 6B</a>. Asterisks indicate the cross-reaction of ORF50 with the used antibodies (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005918#ppat.1005918.s004" target="_blank">S4 Fig</a>). (E) Proposed model for ORF50 ubiquitination. The PARS-I is responsible for the nuclear translocation of ORF50, and the PARS-II motif is required for the binding of specific ubiquitin enzymes. The ubiquitin acceptor sites are likely to be located in the N-terminal 356-aa region.</p

The PARS-II motif functions in the nucleus in the control of ORF50 abundance.

<p>(A) Left, diagram of PARS-I and/or PARS-II mutants of ORF50. A series of ORF50 C-terminal deletions with or without the KK-to-EE mutation in the PARS-I motif were included. All ORF50 mutants contain a FLAG tag at their N terminus. The subcellular localization and protein abundance of each of ORF50 mutants in 293T cells are summarized in the diagram. The degree of protein abundance is indicated by “+”. N: nucleus; C: cytoplasm; N/C: both nucleus and cytoplasm. Right, representative confocal images showing the subcellular localization of these ORF50 mutants. (B) Immunoblot analysis of ORF50 deletion mutants in 293T cells.</p

The protein abundance of ORF50 is controlled through the ubiquitin-proteasomal degradation pathway.

<p>(A) Effect of MG132 on ORF50 abundance in cells. 293T cells were transfected with a plasmid encoding F-ORF50 or F-ORF50(1–564). At 16 hr after transfection, cells were untreated or treated with 5 μM of MG132 for another 12 hr and 24 hr. Cell lysates were analyzed by immunoblotting using anti-FLAG antibody. (B and C) Ubiquitination of ORF50 in cells. 293T cells were transfected with plasmids encoding HA-ubiquitin (HA-Ub) or/and F-ORF50. At 16 hr posttransfection, cells were treated with MG132 for another 24 hr. Denatured lysates were immunoprecipitated (IP) with either anti-HA antibody (B) or anti-FLAG antibody (C). Cell lysates (input) and the immunoprecipitated proteins were analyzed by immunoblotting (IB) using anti-ORF50 or anti-HA antibody. Asterisks indicate the cross-reaction of ORF50 with the used antibodies (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005918#ppat.1005918.s004" target="_blank">S4 Fig</a>).</p

Phenotypic changes of F-ORF50(KK/EE) by different GFP-ORF50 deletion effectors.

<p>(A) Diagram of GFP-ORF50 deletion constructs and a summary of their characteristics. (B) Confocal images of 293T cells coexpressing F-ORF50(KK/EE) and GFP-ORF50 mutants. The subcellular localization of both F-ORF50(KK/EE) (red) and GFP-tagged proteins (green) in transfected cells was analyzed by confocal microscopy. (C) Effect of GFP-ORF50 deletion mutants on the expression of F-ORF50(KK/EE). 293T cells were cotranfected with equal amounts (400 ng) of plasmids encoding F-ORF50(KK/EE) and the indicated GFP-tagged proteins. Cell lysates were immunoblotted with either anti-FLAG antibody or anti-GFP antibody.</p