Dynamic movement of the Golgi unit and its glycosylation enzyme zones.

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.

PIGB maintains nuclear lamina organization in skeletal muscle of Drosophila.

The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function. Here, we found that PIGB, an enzyme involved in glycosylphosphatidylinositol (GPI) synthesis, is responsible for the homogeneous lamin meshwork in Drosophila. Loss of PIGB resulted in heterogeneous distributions of B-type lamin and lamin-binding proteins in larval muscles. These phenotypes were rescued by expression of PIGB lacking GPI synthesis activity. The PIGB mutant exhibited changes in lamina-associated domains that are large heterochromatic genomic regions in the NL, reduction of nuclear stiffness, and deformation of muscle fibers. These results suggest that PIGB maintains the homogeneous meshwork of the NL, which may be essential for chromatin distribution and nuclear mechanical properties.

Hrd1-dependent Degradation of the Unassembled PIGK Subunit of the GPI Transamidase Complex.

Glycosylphosphatidylinositol (GPI)-anchored proteins are post-transcriptionally modified with GPI and anchored to the plasma membrane. GPI is attached to nascent proteins in the endoplasmic reticulum by the GPI transamidase complex, which consists of PIGT, PIGK, GPAA1, PIGU, and PIGS. Of these, PIGK is a catalytic subunit that is unstable without PIGT. This study investigated the pathway by which unassembled PIGK not incorporated into the complex is degraded. We showed that unassembled PIGK was degraded via the proteasome-dependent pathway and that Hrd1 (also known as SYVN1), a ubiquitin ligase involved in the endoplasmic reticulum-associated degradation pathway, was responsible for degradation of unassembled PIGK.Key words: Glycosylphosphatidylinositol, GPI transamidase complex, protein stability, transamidation, ERAD.

SPPL3-dependent downregulation of the synthesis of (neo)lacto-series glycosphingolipid is required for the staining of cell surface CD59.

CD59 is a small glycoprotein modified with a glycophosphatidylinositol (GPI) anchor that prevents the formation of the membrane attack complex, thereby protecting host cells from lysis. A previous study identified that cell surface CD59 staining required the intramembrane protease signal peptide peptidase-like 3 (SPPL3). However, the effect of SPPL3 on the staining of CD59 remains unknown. This study shows that SPPL3 is essential for the surface labeling of CD59 but not of major GPI-anchored proteins. Surface CD59 staining requires the intramembrane protease activity of SPPL3 and SPPL3-mediated suppression of the (neo)lacto-series glycosphingolipids (nsGSLs)-but not N-glycan-synthesis pathway. The abundance of nsGSLs may affect complement-dependent cytotoxicity by altering the abundance or accessibility of cell surface CD59.

Retrolaminar Block Versus Paravertebral Block for Pain Relief After Less-Invasive Lung Surgery: A Randomized, Non-Inferiority Controlled Trial.

Introduction A retrolaminar block (RLB) is a modified paravertebral technique with a local anesthetic injected at the retrolaminar site. The aim of this non-inferiority, parallel-group, prospective, and randomized study was to compare the analgesic efficacy of the paravertebral block (PVB) and RLB after lung surgery. Methods Eligible subjects were patients aged more than 20 years, with American Society of Anesthesiologists physical status Ⅰ or II, who were scheduled to undergo video-assisted thoracoscopic surgery (VATS) or limited thoracotomy because of lung disease. Patients were randomly allocated to receive either a PVB or RLB using a computer-generated sequence and sealed opaque envelopes. The PVB and RLB were induced by injecting 20 mL of 0.50% ropivacaine and 40 mL 0.25% ropivacaine, respectively. As the primary outcome variable, we considered the area under the curve (AUC) of the postoperative pain intensity using the trapezoidal method. Pain intensity was assessed using an 11-point numerical rating scale (NRS). We converted the NRS (0-10) into the visual analog scale (VAS) (0-100 mm) proportionally. We compared the AUC of the converted NRS (AUC-cNRS) on coughing between one and two hours after the operation. The non-inferiority margin was set at 25 mm × h in the AUC-cNRS. Patients and nurses were blinded to group assignments. Secondary outcomes included time to perform the block, NRS for pain intensity at rest and on coughing at one, two, four, 24, and 48 hours after the operation, the incidence of postoperative nausea and vomiting, time to first morphine use after the operation, and cumulative morphine consumption at 24 and 48 hours after the operation. Results In each group, 25 patients were randomized and analyzed. No significant difference in the AUC-cNRS was noted between the groups (P = 0.117). The mean difference in the AUC-cNRS (group RLB minus group PVB) was 13.42 mm × h, 95% confidence interval, -3.48 to 30.32 mm × h. However, when patients with unexpectedly extended skin incision were excluded from the analysis, the AUC-cNRS of group RLB was significantly higher as compared to group PVB (P = 0.0388). The time to perform the block was longer in PVB as compared to the RLB group (P < 0.0001). No significant differences were noted in the remaining secondary outcomes. Conclusion The non-inferiority of RLB as compared to PVB was not confirmed. Though RLB has the advantage of a shorter time to perform, RLB is not recommended for patients undergoing VATS or limited thoracotomy because of lack of efficacy as compared to PVB.

Subunits of the GPI transamidase complex localize to the endoplasmic reticulum and nuclear envelope in Drosophila.

A total of 10-20% of plasma membrane proteins are anchored by glycosylphosphatidylinositol (GPI). GPI is attached to proteins by GPI transamidase (GPI-T), which contains five subunits named PIGK, PIGS, PIGT, PIGU, and GPAA1. We previously reported that PIGT localizes near the nucleus in Drosophila. However, localizations of the other four subunits remain unknown. Here, we show that a catalytic subunit of GPI-T, PIGK, mainly localizes to the endoplasmic reticulum (ER), while the other four subunits localize to the nuclear envelope (NE) and ER. The NE/ER localization ratio of PIGS differs between cell types and developmental stages. Our results suggest that GPI-T catalyzes GPI attachment in the ER and the other four subunits may have other unknown functions in the NE.

Lamin is essential for nuclear localization of the GPI synthesis enzyme PIG-B and GPI-anchored protein production in Drosophila.

Membrane lipid biosynthesis is a complex process that occurs in various intracellular compartments. In Drosophila, phosphatidylinositol glycan-B (PIG-B), which catalyzes addition of the third mannose in glycosylphosphatidylinositol (GPI), localizes to the nuclear envelope (NE). Although this NE localization is essential for Drosophila development, the underlying molecular mechanism remains unknown. To elucidate this mechanism, we identified PIG-B-interacting proteins by performing immunoprecipitation followed by proteomic analysis. We then examined which of these proteins are required for the NE localization of PIG-B. Knockdown of Lamin Dm0, a B-type lamin, led to mislocalization of PIG-B from the NE to the endoplasmic reticulum. Lamin Dm0 associated with PIG-B at the inner nuclear membrane, a process that required the tail domain of Lamin Dm0. Furthermore, GPI moieties were distributed abnormally in the Lamin Dm0 mutant. These data indicate that Lamin Dm0 is involved in the NE localization of PIG-B and is required for proper GPI-anchor modification of proteins.

Stability of the transamidase complex catalyzing GPI anchoring of proteins.

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors some proteins to the plasma membrane. This anchoring is catalyzed by a transamidase complex (TAC) composed of five subunits: PIG-K, GAA1, PIG-U, PIG-T, and PIG-S (Fig. 1A). PIG-K and GAA1 are predicted to catalyze the first and second steps during attachment of proproteins of GPI-anchored proteins (GPI-APs) to GPI. GPI may be delivered by PIG-U, and PIG-T is required for stability of all TAC subunits when overexpressed in cultured cells. However, protein stability of TAC has not been analyzed using loss-of-function mutants for each subunit. Herein, we analyzed the stability of TAC in knockout and/or knockdown mutants for each subunit. PIG-T and PIG-U, or PIG-T and GAA1, were mutually required for stability, and all three subunits were stable without PIG-S or PIG-K. However, these three subunits were essential for the stability of both PIG-S and PIG-K. By contrast, loss of PIG-S reduced the stability of PIG-K and left the other subunits unaffected. Reduction of PIG-K did not impact any of the other subunits. Thus, PIG-T, PIG-U, and GAA1 may form a core complex associated by PIG-S, and these four subunits may stabilize PIG-K, triggering GPI anchoring reactions. Instability of PIG-K in the absence of the other four subunits may ensure that GPI anchoring is catalyzed only by the completely assembled complex.

Nuclear envelope localization of PIG-B is essential for GPI-anchor synthesis in Drosophila.

Membrane lipid biosynthesis is a complex process that takes place in various intracellular compartments. Glycosylphosphatidylinositol (GPI), a lipid involved in membrane anchoring of some proteins, is synthesized by the PIG enzymes. Most PIGs are localized to the endoplasmic reticulum (ER), but Drosophila PIG-B (DmPIG-B) is localized to the nuclear envelope (NE). To determine whether the NE localization of DmPIG-B is functionally important, we defined the determinants of localization and generated an ER-localized form, denoted DmPIG-B[ER]. The enzymatic activity of DmPIG-B[ER] was comparable to that of NE-localized DmPIG-B[NE]. Expression of DmPIG-B[ER] inefficiently rescued the lethality of the PIG-B mutant, whereas DmPIG-B[NE] rescued this lethality fully. DmPIG-B[ER] was preferentially degraded by lysosomes, suggesting that the NE localization is essential for function and stability of the protein. In addition, we found that the region of the ER proximal to the NE is the site of translation of GPI-anchored proteins and addition of GPI. Thus, the NE and proximal ER may provide a platform for efficient GPI anchoring.

[A Case of Aortic Cannula Malposition Detected by Prolonged Desaturation of Regional Cerebral Oxygen Index (rcSO2)].

A sixteen-year-old female patient with congenital aortic stenosis underwent Ross procedure. We monitored bilateral regional cerebral saturation of oxygen (rcSO2) on the forehead at the right and left of the midline. After aortic and bicavel cannulation, cardiopulmonary bypass was instituted. On the mor- row of aortic cross clamping, the rcSO2 fell from approximately 55% to below 30%. We searched the cause of this phenomenon, and detected that the tip of aortic cannula was inserted to the left subclavian artery. After repositioning, the bilateral rcSO2 increased to above 65%. We felt keenly that the monitoring of rcSO2 is useful to recognize corrective adjustment of the cannula ori- entation, and the avoidance of cerebral hypoperfusion during the cardiopulmonary bypass period.

[Catecholamine Cardiomyopathy Presenting Inverted-takotsubo Pattern Asynergy].

Pheochromocytoma is complicated with catecholamine cardiomyopathy, and preoperative care becomes considerably more difficult We report anesthetic management for an 48-year-old man brought to our hospital by ambulance and immediately hospitalized due to pheochromocytoma crisis complicated with catechol- amine cardiomyopathy presenting inverted-takotsubo pattern asynergy. Before surgery, α and ß blocking drugs were used to control hypertension and tachycardia, and continuous hemodiafiltration was initiated and the patient was intubated for severe pulmonary edema. Seven days later, he underwent laparoscopic adrenalectomy. Total intravenous anesthesia was achieved with propofol, remifentanil, fentanyl, and rocuronium. During the operation, continuous infusion of landiolol, phentolamine, and nitroglycerin, and addi- tional bolus injections of landiolol were given to control hypertension. After severing the right adrenal vein, noradrenaline (0.15 µg · kg⁻¹ · min⁻¹), and dopamine (4µg · kg⁻¹ · min⁻¹) were started and the patient was placed in the intensive care unit Inverted-takotsubo pattern asynergy is not very common, and treatment consists of supportive care, as in the usual takotsubo. Preoperative CHDF (continuous hemodiafiltration) may be useful when it is difficult to control hypertension or tachycardia with medications.

[General Anesthesia for Septal Myectomy in a Patient with Noonan Syndrome, Severe Hypertrophic Obstructive Cardiomyopathy and Right Ventricular Outflow Tract Obstruction].

We report a case of an 18-year-old man with Noonan syndrome, severe hypertrophic obstructive cardiomy- opathy and right ventricular outflow tract obstruction who underwent septal myectomy under general anes- thesia. In our case, the ventricular outflow tract pres- sure gradients were 108 mmHg at left and 79 mmHg at right. General anesthesia was induced deliberately by fentanyl, midazolam and sevoflurane. Anesthesia was maintained with sevoflurane. For treatment of hypotension, we performed volume loading and admin- istration of phenylephrine. We did not use drugs that increase heart rate or contractility. Preload and after- load were well maintained, and the operation and gen- eral anesthesia were completed without serious prob- lem.

Spätzle-Processing Enzyme-independent Activation of the Toll Pathway in Drosophila Innate Immunity.

The Toll pathway regulates innate immunity in insects and vertebrates. The Drosophila Toll receptor is activated by a processed form of a ligand, Spätzle. Spätzle-processing enzyme (SPE) is the only enzyme identified to date that functions in converting Spätzle to an active form during the immune response. In the present study, Toll activation induced by immune challenge was almost suppressed in spätzle mutant larvae and adults, whereas it was present in SPE mutant larvae challenged with Micrococcus luteus and adults challenged with Bacillus subtilis. Our data suggest that an unidentified protease besides SPE processes Spätzle under conditions of microbial challenge.

[Anesthetic Management of Single-staged Definitive Repair of Pulmonary Atresia with Ventricular Septum Defect and Major Aorto-pulmonary Collateral Arteries (PA/VSD, MAPCA)in an Adult].

A 25-year-old woman with unrepaired pulmonary atresia, ventricular septal defect and major aorto-pul- monary collateral artery was scheduled for single- staged definitive repair. She was complicated with mod- erate pulmonary hypertension, and had 2 MAPCAs arising from the descending artery. Cardiac catheter- ization demonstrated that right ventricular pressures were approximately equal to the left ventricular pres- sure. Pre-bypass, we maintained her PVR not too high in order to maintain her pulmonary blood flow. Post- bypass, we used hemodynamic support with dopamine, olprinone and nitroglycerin, maintaining her PVR opti- mally low. We inserted a catheter introducer in her jugular vein to deal with massive hemorrhage. After the repair, her right ventricular pressures were 7/10 of systemic pressure, and her postoperative course was uneventful.

Phenotype-based clustering of glycosylation-related genes by RNAi-mediated gene silencing.

Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, ß1,4-galactosyltransferase I, ß1,3-galactosyltransferase II, and ß1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.

Dynamic regulation of innate immune responses in Drosophila by Senju-mediated glycosylation.

The innate immune system is the first line of defense encountered by invading pathogens. Delayed and/or inadequate innate immune responses can result in failure to combat pathogens, whereas excessive and/or inappropriate responses cause runaway inflammation. Therefore, immune responses are tightly regulated from initiation to resolution and are repressed during the steady state. It is well known that glycans presented on pathogens play important roles in pathogen recognition and the interactions between host molecules and microbes; however, the function of glycans of host organisms in innate immune responses is less well known. Here, we show that innate immune quiescence and strength of the immune response are controlled by host glycosylation involving a novel UDP-galactose transporter called Senju. In senju mutants, reduced expression of galactose-containing glycans resulted in hyperactivation of the Toll signaling pathway in the absence of immune challenges. Genetic epistasis and biochemical analyses revealed that Senju regulates the Toll signaling pathway at a step that converts Toll ligand Spatzle to its active form. Interestingly, Toll activation in immune-challenged wild type (WT) flies reduced the expression of galactose-containing glycans. Suppression of the degalactosylation by senju overexpression resulted in reduced induction of Toll-dependent expression of an antimicrobial peptide, Drosomycin, and increased susceptibility to infection with Gram-positive bacteria. These data suggest that Senju-mediated galactosylation suppresses undesirable Toll signaling activation during the steady state; however, Toll activation in response to infection leads to degalactosylation, which raises the immune response to an adequate level and contributes to the prompt elimination of pathogens.

In Vivo RNAi-Based Screens: Studies in Model Organisms.

RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.

Balanced ubiquitination determines cellular responsiveness to extracellular stimuli.

Signal strength evoked by ligand stimulation is crucial for cellular responses such as fate decision, cell survival/death, secretion, and migration. For example, morphogens are secreted signaling molecules that form concentration gradients within tissues and induce distinct cell fates in a signal strength-dependent manner. In addition to extracellular ligand abundance, the sensitivity of signal-receiving cells to ligands also influences signal strength. Cell sensitivity to ligands is controlled at various levels: receptor presentation at the cell surface, positive/negative regulation of signal transduction, and target gene activation/repression. While the regulation of signal transduction and gene transcription is well studied, receptor presentation is still not fully understood. Recently, it was reported that cellular sensitivity to the Wingless (Wg)/Wnt morphogen is regulated by balanced ubiquitination and deubiquitination of its receptor Frizzled (Fz). In this review, we review how ubiquitination regulates receptor presentation at the cell surface for the detection of extracellular signal strength.

Identification of proteasome components required for apical localization of Chaoptin using functional genomics.

Abstract: the distinct localization of membrane proteins with regard to cell polarity is crucial for the structure and function of various organs in multicellular organisms. However, the molecules and mechanisms that regulate protein localization to particular subcellular domains are still largely unknown. To identify the genes involved in regulation of protein localization, the authors performed a large-scale screen using a Drosophila RNA interference (RNAi) library, by which Drosophila genes could be knocked down in a tissue- and stage-specific manner. Drosophila photoreceptor cells have a morphologically distinct apicobasal polarity, along which Chaoptin (Chp), a glycosylphosphatidylinositol (GPI)-anchored membrane protein, and the Na (+) , K(+) -ATPase are localized to the apical and basolateral domains, respectively. By examining the subcellular localization of these proteins, the authors identified 106 genes whose knockdown resulted in mislocalization of Chp and Na(+) , K(+) -ATPase. Gene ontology analysis revealed that the knockdown of proteasome components resulted in mislocalization of Chp to the basolateral plasma membrane. These results suggest that the proteasome is involved, directly or indirectly, in selective localization of Chp to the apical plasma membrane of Drosophila photoreceptor cells.

Cisterna-specific localization of glycosylation-related proteins to the Golgi apparatus.

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.