Gen Matsuura

Two Distinct Cytotoxic Activities of Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli

ABSTRACT Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 μg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was ∼1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.

Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin

Some shiga toxin‐producing Escherichia coli secrete a novel AB5 cytotoxin, named subtilase cytotoxin (SubAB), which induces vacuole formation in addition to cytotoxicity in susceptible cells. By immunoprecipitation with SubAB from Vero cells, we discovered proteins of 100 kDa, 135 kDa and 155 kDa as potential candidates for its receptor. These proteins were N‐glycosylated in their extracellular domains, a modification that was necessary for interaction with SubAB. Biotinylated receptors were partially purified by Datura stramonium agglutinin affinity chromatography and avidin‐agarose and analysed by TOF mass spectroscopy. The peptide sequences of p135 were identical to β1 integrin, and its identification was confirmed with anti‐integrin β1 antibody. The p155 protein was identified as α2 integrin using anti‐integrin α2 antibody. In addition, treatment of Vero cells with β1 integrin RNAi before exposure to SubAB prevented vacuolating activity. These results suggested that SubAB recognizes α2β1 integrin as a functional receptor; this first interaction may be an important key step leading to the SubAB‐induced morphological changes in Vero cells.

Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1

Subtilase cytotoxin (SubAB) is a AB5 type toxin produced by Shiga‐toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase‐like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double‐stranded RNA‐activated protein kinase‐like ER kinase (PERK) and eukaryotic initiation factor‐2α (eIF2α). The phosphorylation of PERK and eIF2α was maximal at 30–60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress‐inducible C/EBP‐homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB‐treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB‐induced translational inhibition and continuous prolonged proteasomal degradation.

Novel Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli Induces Apoptosis in Vero Cells via Mitochondrial Membrane Damage

ABSTRACT Subtilase cytotoxin (SubAB) is an AB5 cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G1 phase. Here we show that SubAB, but not the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA fragmentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cytochrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.

Use of preoperative, 3-dimensional magnetic resonance cholangiopancreatography in pediatric choledochal cysts

BACKGROUND Standard choledochal cyst (CC) operations involve dilated extrahepatic bile duct excision followed by biloenterostomy. However, biliary variants and associated intrahepatic bile duct (IHBD) stenoses or dilatations triggering postoperative sequelae require additional procedures. The usefulness of preoperative 3-dimensional magnetic resonance cholangiopancreatography (3D MRCP) and virtual cholangioscopy (VES) for observing biliary morphology and pancreaticobiliary maljunction (PBM) was evaluated. METHODS In 16 pediatric CC patients (age range, 4 months to 9 years; median, 3 years), visualization of PBM and aberrant bile duct anatomy and IHBD morphology at the hepatic hilum (HH), umbilical portion (UP), and posterior branch (POST) were compared between 3D-MRCP and intraoperative cholangiography (IOC). VES and intraoperative cholangioscopy (IOS) findings were compared. RESULTS HH, UP, and POST visualization rates were 100%, 94%, and 94%, respectively, by 3D-MRCP, and 100%, 69%, and 69%, respectively, by IOC. IHBD stenosis detection rates at each region were 38%, 13%, and 13%, respectively, by 3D-MRCP, and 25%, 0%, and 9%, respectively, by IOC. IHBD dilatation detection rates at each part were 75%, 47%, and 60%, respectively, by 3D-MRCP, and 88%, 82%, and 91%, respectively, by IOC. PBM was confirmed in 56% and 93% of cases on 3D-MRCP and IOC, respectively. Both 3D-MRCP and IOC showed biliary variants in 5 cases (31%). VES showed membranous strictures at HH, UP, and POST in 6, 2, and 2 cases, respectively, whereas IOS did so at HH in 4 cases and POST in 2. CONCLUSION Preoperative 3D-MRCP and VES accurately depict biliary morphology, allowing concrete operative planning in pediatric CC patients, complementing IOC and IOS.

A rare presentation in a case of gastric duplication cyst communicating to the pancreatic duct: coincidental detection during pyloromyotomy for hypertrophic pyloric stenosis

A rare case of gastric duplication of childhood is reported. A 1-month-old boy underwent pyloromyotomy for infantile hypertrophic pyloric stenosis. At laparotomy, a protruding lesion was identified in the anterior wall of the pylorus. The pylorus was hypertrophic as indicated preoperatively, and a normal pyloromyotomy was performed. Wedge biopsy of the protruding lesion revealed an intestinal structure, and gastric duplication was diagnosed. The child remained asymptomatic thereafter, but considering the risk of the duplication becoming symptomatic, a second laparotomy was performed at 1 year of age. The duplication cyst shared part of the wall with the pylorus, and the cyst was removed by cystectomy, leaving the pyloric muscle intact. The cyst was found to be communicating with the pancreatic duct. The child is doing well without any symptoms as of 18 months after second laparotomy. To the best of our knowledge, this is the youngest case of gastroduodenal duplication with pancreatic duct communication yet reported.

Extirpation of mass-screened adrenal neuroblastomas by retroperitoneoscopy

Background: A mass-screened neuroblastoma (MSN) shows low-grade malignancy. Although the tumor is small, open laparotomy requires a large incision. Purpose: Extirpation by retroperitoneoscopy (ER) was performed in children with MSN to reduce the surgical stress. Methods: The indications for ER were (1) MSN originating from the adrenal gland and (2) a tumor size of less than 4 cm. Between January 2000 and June 2002, 6 children (mean age: 8.5 ± 0.6 months) underwent ER. Results: All children successfully underwent ER and no conversion of laparoscopy to open surgery was required. The only complication in one patient was a peritoneal tear. Oral intake was possible in all children the day after the operation. No port site metastasis or local recurrence was observed during follow-up (15 to 29 months). Conclusions: Adrenal MSN less than 4 cm were successfully extirpated by ER. This method has the advantage of allowing patients an early return to eating and has no risk of postoperative ileus. ER is an available surgical technique for children with MSN less than 4 cm.

Efficacy of ERCP via the accessory papilla in children with choledochal cysts.

BACKGROUND ERCP via the major duodenal papilla in children with choledochal cyst may not clearly visualize the entire pancreatic duct and the junction of the pancreatic and biliary ducts. This may be caused by obstruction of the pancreatic duct by a dilated common bile duct. METHODS Patients with choledochal cysts who underwent ERCP with injection of contrast medium at the major duodenal papilla were classified as either belonging to a MP group, in which the entire pancreatic duct and junction of the pancreatic and biliary ducts were visualized, or to a M/AP group, in which these structures were either partially visualized or not visualized. ERCP via the accessory papilla subsequently was performed in the M/AP group by using 0.6- or 0.8-mm metal-tip catheters. The efficacy of ERCP via the accessory papilla in children with choledochal cysts was evaluated. OBSERVATIONS ERCP was performed in 13 patients. Seven were assigned to the MP group and 6 to the M/AP group. ERCP via the accessory papilla in the M/AP group was successful in 5 of the 6 patients; pancreas divisum was demonstrated in one, a protein plug at the main pancreatic duct in another, and the entire pancreatic duct and junction of the pancreatic and biliary ducts in the remaining 3 patients. No patient developed pancreatitis as a result of ERCP, including ERCP via the accessory papilla. CONCLUSIONS In children with choledochal cyst, ERCP via the accessory papilla is an effective method for visualization of the detailed structure of the entire pancreatic ductal system and junction of the pancreatic and biliary ducts when ERCP via the major duodenal papilla is unsuccessful.

Diagnosis of adrenocortical tumor in a neonate by detection of elevated blood 17-hydroxyprogesterone measured as a routine neonatal screening for congenital adrenal hyperplasia: a case report

We report herein a case of prenatally detected neonatal adrenocortical tumor (ACT). The patient was an otherwise healthy newborn girl. No signs of Beckwith-Wiedemann syndrome were identified, and her family medical history did not suggest predisposition to cancer. Computed tomography and ultrasonography after birth revealed a round solid tumor 40 mm in diameter in the right suprarenal area. The precise diagnosis of ACT was unexpectedly obtained based on results from the Japanese neonatal mass screening program. Blood 17-hydroxyprogesterone is routinely measured as a part of this program for early detection of congenital adrenal hyperplasia in Japan. Abnormally elevated level of 17-hydroxyprogesterone was reported in the patient and, thus, led to the diagnosis of ACT. Surgical resection was safely performed with perioperative steroid replacement. Adrenocortical tumors are extremely rare in childhood, particularly in the neonatal period. Some of these tumors secrete abnormally high levels of cortisol, suppressing function of the contralateral adrenal gland and, thus, leading to life-threatening postoperative adrenal insufficiency. Scheduled steroid replacement enables safe perioperative management in such cases. Adrenocortical tumor should always be considered among the differential diagnoses for neonatal suprarenal mass because precise diagnosis will enable the physician to develop appropriate treatment strategies.

Prediction of respiratory collapse among pediatric patients with mediastinal tumors during induction of general anesthesia

PURPOSE Fatality resulting from respiratory collapse (RC) during general anesthesia (GA) induction in children with mediastinal tumors has been reported. We explored potentially useful parameters for predicting the risk of RC based on objective imaging results. METHODS We retrospectively reviewed the records of 31 patients (median age: 4years; range: 4months-15years) with mediastinal tumors treated between 2000 and 2015. Comparing those with RC under GA induction to those without (RC group vs. non-RC group), we analyzed a variety of factors that might predict respiratory obstruction during GA induction, including our new parameter, the standardized tumor volume (STV), which is adapted from the formula for the volume of an ellipsoid. RESULTS All eight patients in the RC group had large tumors in the anterior mediastinum, including lymphoma, teratoma, and germ cell tumor. The mean STV value of the RC group was significantly larger than that of the non-RC group (3.6±1.4 vs. 1.4±1.0, p=0.006). Using an STV cut-off value of 2.5, the sensitivity and specificity for predicting RC under GA induction were both 0.86, making STV more useful than previously reported risk factors. CONCLUSION Anterior mediastinal tumors in children can often cause airway obstruction under GA induction. Measuring STV can help predict the respiratory risk during GA among pediatric patients with anterior mediastinal tumors. LEVEL OF EVIDENCE III.