Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

Coordinated regulation of TORC2 signaling by MCC/eisosome-associated proteins, Pil1 and tetraspan membrane proteins during the stress response.

MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC function. Pil1 is a core eisosome protein and is responsible for MCC-invaginated structures. In addition, six-tetraspan membrane proteins (6-Tsp) are localized in the MCCs and classified into two families, the Sur7 family and Nce102 family. To understand the coordinated function of these MCC proteins, single and multiple deletion mutants of Pil1 and 6-Tsp were generated and their MCC structure and growth under various stresses were investigated. Genetic interaction analysis revealed that the Sur7 family and Nce102 function in stress tolerance and normal eisosome assembly, respectively, by cooperating with Pil1. To further understand the role of MCCs/eisosomes in stress tolerance, we screened for suppressor mutants using the SDS-sensitive phenotype of pil1Δ 6-tspΔ cells. This revealed that SDS sensitivity is caused by hyperactivation of Tor kinase complex 2 (TORC2)-Ypk1 signaling. Interestingly, inhibition of sphingolipid metabolism, a well-known downstream pathway of TORC2-Ypk1 signaling, did not rescue the SDS-sensitivity of pil1Δ 6-tspΔ cells. These results suggest that Pil1 and 6-Tsp cooperatively regulate TORC2 signaling during the stress response.

Regulation of sphingolipid biosynthesis in the endoplasmic reticulum via signals from the plasma membrane in budding yeast.

Saccharomyces cerevisiae LIP1 encodes a regulatory subunit that forms a complex with the ceramide synthase catalytic subunits, Lag1/Lac1, which is localized on the membrane of endoplasmic reticulum. To understand the underlying regulatory mechanism of sphingolipid biosynthesis, we generated strains upon replacing the chromosomal LIP1 promoter with a Tet-off promoter, which enables the expression in Dox-dependent manner. The lip1-1 strain, obtained through the promoter substitution, exhibits severe growth inhibition and remarkable decrease in sphingolipid synthesis in the presence of Dox. Using this strain, we investigated the effect of a decrease in ceramide synthesis on TOR complex 2 (TORC2)-Ypk1 signaling, which senses the complex sphingolipid level at the plasma membrane and promotes sphingolipid biosynthesis. In lip1-1 cells, Ypk1 was activated via both upstream kinases, TORC2 and yeast PDK1 homologues, Pkh1/2, thereby inducing hyperphosphorylation of Lag1, but not of another Ypk1-substrate, Orm1, which is a known negative regulator of the first step of sphingolipid metabolism, in the presence of Dox. Therefore, our data suggest that the metabolic enzyme activities at each step of the sphingolipid biosynthetic pathway are controlled through a fine regulatory mechanism.

Acromioclavicular Joint Stabilization: A Biomechanical Study of Bidirectional Stability and Strength.

BACKGROUND: It is important to restore horizontal and vertical stability to the acromioclavicular (AC) joint when treating dislocations of this joint. Most surgical stabilization techniques of the AC joint have primarily addressed the coracoclavicular ligament complex; however, these techniques may not satisfactorily restore horizontal stability to the AC joint. PURPOSE: To evaluate the strength and bidirectional stability of 3 AC joint stabilizing techniques in a cadaveric model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 24 cadaveric shoulders were randomly allocated to 3 treatment groups. For each group, a standardized AC joint stabilizing procedure was performed, and the specimens were potted for mechanical testing. The following reconstruction techniques were used: a single clavicular tunnel for group A, a double clavicular tunnel for group B, and a double clavicular tunnel plus suture fixation across the AC joint for group C. The specimens underwent cyclic loading in the horizontal and vertical planes and then load to failure. Eight control specimens also underwent cyclic loading in both planes. Construct stiffness during cyclic loading, change in displacement after cyclic loading in both planes, load to failure in the vertical plane, and mode of failure were evaluated, and stiffness was compared among the treatment groups as well as with a control group. RESULTS: There was a decrease in joint stiffness for all groups, including controls, during the cyclic loading. Compared with controls, all 3 treatment groups demonstrated equivalent stiffness and displacement in the vertical plane. In the horizontal plane, all 3 treatment groups demonstrated decreased stiffness, increased displacement, or both when compared with controls. When groups were compared, no treatment arm proved superior regarding stiffness or displacement in either plane. Load-to-failure testing of the 3 treatment groups in the vertical plane demonstrated construct strength and stiffness comparable with reports for the native AC joint. The mode of failure was predominantly fracture at the point of fixation to the testing apparatus. CONCLUSION: There was no difference in bidirectional strength and stability between the single- and double-clavicular tunnel techniques of coracoclavicular reconstruction. The addition of a stabilizing suture across the AC joint does not improve horizontal stability in the absence of repair of the AC joint capsule and deltotrapezial fascia. CLINICAL RELEVANCE: This laboratory study provides further evidence of the importance of the AC joint capsule and associated soft tissues in affording horizontal stability to that joint. Information from this and subsequent studies utilizing a bidirectional model can influence the choice of surgical procedure in the clinical treatment of AC joint dislocations.

Perinatal exposure to tetrabromobisphenol A (TBBPA), a brominated flame retardant, exacerbated the pneumonia in respiratory syncytial virus (RSV)-infected offspring mice.

To investigate the effects of perinatal exposure to tetrabromobisphenol A (TBBPA), a brominated flame retardant, on the immune system, a respiratory syncytial virus (RSV) infection mouse model was utilized. Female mice were exposed to TBBPA mixed with the diet from 10 days after conception to weaning on postnatal day 21. Offspring mice were infected intranasally with A2 strain of RSV. Although no general toxicological sign was observed, the pulmonary viral titers of offspring mice exposed to 0.1% TBBPA were significantly increased compared with the control on day 5 post-infection. TBBPA did not affect RSV growth in vitro. Histopathological analysis confirmed that the exacerbation of interstitial pneumonia was due to TBBPA- exposure in the lung tissues in RSV-infected offspring. Moreover, gene expression of interleukin (IL)-24 was shown to be elevated typically in the lung tissues of TBBPA-treated offspring by a DNA microarray and was also confirmed by immunohistopathological analysis using an anti-IL-24 antibody. Thus, developmental exposure to TBBPA affected the immune response to RSV infection, resulting in the exacerbation of pneumonia. Thus, IL-24 should be a key molecule to understand the mechanism of action of TBBPA.

Endoscopic management of post-cholecystectomy biliary fistula.

OBJECTIVE: Bile duct injury is an uncommon but potentially serious complication in cholecystectomy. A recognized treatment for minor biliary injury is internal biliary decompression by endoscopic retrograde cholangiopancreatography (ERCP) and stent insertion. The aim of this study was to assess the effectiveness of ERCP in the management of minor biliary injuries. METHODS: A retrospective review of medical records at a tertiary referral centre identified 36 patients treated for postoperative minor biliary injuries between 2006 and 2010. Management involved establishing a controlled biliary fistula followed by ERCP to confirm the nature of the injury and decompress the bile duct with stent insertion. RESULTS: Controlled biliary fistulae were established in all 36 patients. Resolution of the bile leak was achieved prior to ERCP in seven patients, and ERCP with stent insertion was successful in 27 of the remaining 29 patients. Resolution of the bile leak was achieved in all patients without further intervention. The median time to resolution after successful ERCP was 4 days. Two patients underwent ERCP complicated by mild pancreatitis. No other complications were seen. CONCLUSIONS: This review confirms that postoperative minor biliary injuries can be managed by sepsis control and semi-urgent endoscopic biliary decompression.