Minimal endoscopic sphincterotomy followed by papillary balloon dilation to relieve choledocholithiasis in a 6-year-old girl with hereditary spherocytosis.

A 6-year-old girl previously diagnosed with hereditary spherocytosis was admitted to our hospital with gallstones and cholangitis. Endoscopic retrograde cholangiopancreatography (ERCP) was performed, and fluoroscopy revealed a dilated common bile duct (CBD) without evident stones, possibly due to spontaneous excretion through the papilla of Vater. A 7-French plastic stent was inserted into the CBD. After the procedure, a marked increase in pancreatic enzyme levels was observed, and she was diagnosed with post-ERCP pancreatitis (PEP). Stent placement could have been a cause of pancreatitis; therefore, we removed the stent. Subsequently, recovery from pancreatitis was confirmed, although she suddenly complained of abdominal pain and was diagnosed with choledocholithiasis recurrence. ERCP was repeated, and fluoroscopy revealed a dilated CBD with a stone. A minimal endoscopic sphincterotomy (EST) was performed to reduce the risk of PEP, and a biliary dilation balloon placed across the papilla was gradually inflated until the waist of the balloon disappeared. Stones were extracted using a retrieval balloon catheter. The abdominal pain resolved immediately, and the patient recovered without developing PEP. To our knowledge, this is the first case report of a pediatric patient treated with minimal EST followed by papillary balloon dilation for choledocholithiasis.

Evaluation of the mechanical properties of current biliary self-expandable metallic stents: axial and radial force, and axial force zero border.

BACKGROUND/AIMS: Mechanical properties (MPs) and axial and radial force (AF and RF) may influence the efficacy and complications of self-expandable metallic stent (SEMS) placement. We measured the MPs of various SEMSs and examined their influence on the SEMS clinical ability. METHODS: We evaluated the MPs of 29 types of 10-mm SEMSs. RF was measured using a conventional measurement device. AF was measured using the conventional and new methods, and the correlation between the methods was evaluated. RESULTS: A high correlation in AFs was observed, as measured by the new and conventional manual methods. AF and RF scatterplots divided the SEMSs into three subgroups according to structure: hook-and-cross-type (low AF and RF), cross-type (high AF and low RF), and laser-cut-type (intermediate AF and high RF). The hook-and-cross-type had the largest axial force zero border (>20°), followed by the laser-cut and cross types. CONCLUSION: MPs were related to stent structure. Hook-and-cross-type SEMSs had a low AF and high axial force zero border and were considered safest because they caused minimal stress on the biliary wall. However, the increase in RF must be overcome.

Efficacy and Safety of Peroral Pancreatoscopy Through the Fistula Created by Endoscopic Ultrasound-Guided Pancreaticogastrostomy.

OBJECTIVES: Endoscopic ultrasound/endosonography-guided pancreaticogastrostomy (EUS-PGS) is a useful alternative when endoscopic retrograde pancreatography is difficult. Recently, many procedures, including peroral pancreatoscopy (POPS), have been performed through the mature fistula (MF) created by EUS-PGS. We evaluate the efficacy and safety of POPS to diagnose and treat pancreatic ductal stricture/pancreato-jejunal anastomotic stricture (PDS/PJAS) through the MF. METHODS: Twenty patients underwent EUS-PGS; 13 of these underwent POPS through the MF at Juntendo University Hospital. All patients were studied retrospectively in terms of technical and clinical success rates and adverse events (AEs). RESULTS: The technical and clinical success rates of EUS-PGS were 95% and 100%. The early and late AEs rates were 20% and 15%. The technical success rate of POPS was 100%, with one AE. Biopsy of PDS/PJAS under POPS guidance revealed recurrent/residual intrapapillary mucinous adenoma (3 patients) and benign fibrotic stricture (10 patients). In the latter patients, multiple plastic stents were placed to dilate PDS/PJAS. Four patients with improvement of PDS/PJAS were stent free, but the remaining patients were not yet. CONCLUSIONS: Endoscopic ultrasound/endosonography-guided pancreaticogastrostomy and various procedures (including POPS) performed through the MF are feasible and effective and can diagnose and treat PDS/PJAS with acceptable AEs rates.

Impact of the Coronavirus Disease-2019 Pandemic on Pancreaticobiliary Disease Detection and Treatment.

The emergency declaration (ED) associated with the coronavirus disease-2019 (COVID-19) pandemic in Japan had a major effect on the management of gastrointestinal endoscopy. We retrospectively compared the number of pancreaticobiliary endoscopies and newly diagnosed pancreaticobiliary cancers before (1 April 2018 to 6 April 2020), during (7 April to 25 May 2020), and after the ED (26 May to 31 July). Multiple comparisons of the three groups were performed with respect to the presence or absence of symptoms and clinical disease stage. There were no significant differences among the three groups (Before/During/After the ED) in the mean number of diagnoses of pancreatic cancer and biliary cancer per month in each period (8.0/7.5/7.5 cases, p = 0.5, and 4.0/3.5/3.0 cases, p = 0.9, respectively). There were no significant differences among the three groups in the number of pancreaticobiliary endoscopies (EUS: endoscopic ultrasonography/ERCP: endoscopic retrograde cholangiopancreatography) per month (67.8/62.5/69.0 cases, p = 0.7 and 89.8/51.5/86.0 cases, p = 0.06, respectively), whereas the number of EUS cases decreased by 42.7% between before and during the ED. There were no significant differences among the three groups in the presence or absence of symptoms at diagnosis or clinical disease stage. There was no significant reduction in the newly diagnosed pancreaticobiliary cancer, even during the ED. The number of ERCP cases was not significantly reduced as a result of urgent procedures, but the number of EUS cases was significantly reduced.

Usefulness of Routine Plain CT the Day After an Interventional EUS Procedure.

BACKGROUND: Interventional endoscopic ultrasound-guided procedures (I-EUS) are widely accepted as salvage procedures in ERCP-failed cases, and to drain fluid collected in the abdominal cavity. Although I-EUS has a relatively high incidence of complications and is severe/fatal in some cases, no follow-up strategy has been established. In our institution, plain computed tomography (P-CT) is performed routinely the day after I-EUS. In this study, we evaluated the usefulness of routine P-CT the day after I-EUS, as a follow-up method, and propose an algorithm. METHODS: We retrospectively reviewed 81 patients who underwent I-EUS and evaluated the usefulness of P-CT, abdominal X-ray, laboratory data, and symptoms as a follow-up method. An adverse event (AE) was defined as an event requiring any treatment. RESULTS: Technical success, clinical success, and AE rates were 96.3%, 90.1%, and 18.9%, respectively. In total, 30 patients had abnormal findings among the follow-up methods: 6 cases underwent additional procedures, 8 underwent medical treatments, and 16 were observed. The sensitivity, specificity, and accuracy for detecting AEs were assessed based on P-CT (85.7%, 100%, and 97.5%), X-ray (7.1%, 100%, and 83.5%), laboratory data (71.4%, 83.0%, and 81.0%), and symptoms (92.9%, 86.2%, and 87.3%). The sensitivity and accuracy of the latter two items were as high as those for X-ray, but specificity was lower than those for X-ray and P-CT. CONCLUSIONS: Routine P-CT the day after I-EUS was useful for detecting complications and deciding to perform an invasive salvage procedure. Symptoms and laboratory data were useful to supplement routine P-CT.

Duration of Reduced CA19-9 Levels Is a Better Prognostic Factor Than Its Rate of Reduction for Unresectable Locally Advanced Pancreatic Cancer.

A decrease in carbohydrate antigen (CA) 19-9 levels has been proposed as a prognostic marker for survival and recurrence in patients with pancreatic cancer. We evaluated the association between duration of reduced CA 19-9 levels during 6 months after treatment and long-term survival for 79 patients with unresectable locally advanced pancreatic cancer (LAPC). We calculated the differences between pretreatment and monthly CA19-9 levels. We categorized 71 patients with decreases in CA19-9 levels into three groups based on the duration of these reduced levels (>2, >3, and >4 months). The cut-off level for long-term (more than 2 years) survival was identified as a 44% reduction from the baseline, using a ROC curve. A reduction duration >2 months was not associated with overall survival (p = 0.1), while >3 months was significantly associated with survival (p =.04). In multivariate analysis, a reduction duration >3 months predicted a good long-term prognosis (odds ratio = 5.75; 95% confidence interval = 1.47-22.36; p < 0.01). In patients with unresectable LAPC, the duration of reduced CA19-9 levels for more than 3 months, rather than the rate of reduction in CA19-9 levels, during 6 months after treatment was significantly associated with good prognosis.

Risk Factors for Stent Migration into the Abdominal Cavity after Endoscopic Ultrasound-Guided Hepaticogastrostomy.

Background: Endoscopic ultrasound-guided hepaticogastrostomy (EUS-HGS) is becoming increasingly popular. However, the risk factors for stent migration into the abdominal cavity remain unknown. Methods: Forty-eight patients undergoing EUS-HGS with placement of a long, partially covered self-expandable metallic stent (LPC-SEMS) were studied retrospectively to identify risk factors of stent migration. We determined the technical and functional success rates, and recorded adverse events, including stent migration. Results: EUS-HGS was technically successful in all patients. However, stent migration was evident in five patients (one actual and four imminent, 10%). Stent migration into the abdominal cavity was observed in one patient (2%), and the other four cases required additional procedures to prevent migration (8%). Logistic regression analysis revealed that the risk of stent migration increased as the initial (pre-procedure) distance between the stomach and liver at the puncture site increased (p = 0.012). Conclusions: A longer distance between the stomach and liver at the puncture site increased the risk of stent migration. However, during EUS-HGS, it is difficult to adjust the puncture position. It is important to ensure that the proportion of the stent in the stomach is large; the use of a self-anchoring stent may be optimal.

Evaluation of the Feasibility and Effectiveness of Placement of Fully Covered Self-Expandable Metallic Stents via Various Insertion Routes for Benign Biliary Strictures.

BACKGROUND AND AIMS: The goals of the management of benign biliary stricture (BBS) are to relieve symptoms and resolve short-/long-term stricture. We performed fully covered self-expandable metallic stent (hereafter, FCSEMS) placement for BBS using various methods and investigated the treatment outcomes and adverse events (AEs). METHODS: We retrospectively studied patients who underwent FCSEMS placement for refractory BBS through various approaches between January 2017 and February 2020. FCSEMS were placed for 6 months, and an additional FCSEMS was placed if the stricture had not improved. Technical success rate, stricture resolution rate, and AE were measured. RESULTS: A total of 26 patients with BBSs that were difficult to manage with plastic stents were included. The mean overall follow-up period was 43.3 ± 30.7 months. The cause of stricture was postoperative (46%), inflammatory (31%), and chronic pancreatitis (23%). There were four insertion methods: endoscopic with duodenoscopy, with enteroscopy, EUS-guided transmural, and percutaneous transhepatic. The technical success rate was 100%, without any AE. Stricture resolution was obtained in 19 (83%) of 23 cases, except for three cases of death due to other causes. Stent migration and cholangitis occurred in 23% and 6.3%, respectively. Stent fracture occurred in two cases in which FCSEMSs were placed for more than 6 months (7.2 and 10.3 months). CONCLUSION: FCSEMS placement for refractory BBS via various insertion routes was feasible and effective. FCSEMSs should be exchanged every 6 months until stricture resolution because of stent durability. Further prospective study for confirmation is required, particularly regarding EUS-guided FCSEMS placement.

Endoscopic ultrasound-guided antegrade procedures for managing bile duct stones in patients with surgically altered anatomy: Comparison with double-balloon enteroscopy-assisted endoscopic retrograde cholangiography (with video).

BACKGROUND AND STUDY AIM: Management of bile duct stones (BDSs) in patients with surgically altered anatomies (SAAs) remains challenging. An endoscopic ultrasound-guided antegrade (EUS-AG) procedure and double-balloon enteroscopy-assisted endoscopic retrograde cholangiography (DB-ERC) have been used to remove BDSs from patients with SAAs. However, few comparative data have been reported. Therefore, we compared the efficacy and safety of the techniques. METHODS: This was a single-center retrospective study. Patients with SAA who underwent the EUS-AG procedure or DB-ERC to remove intra- or extra-BDSs between November 2010 and March 2020 were included. The primary outcome was the technical success rate, defined as stent insertion or stone removal during the initial session. The secondary outcomes were the procedure time, incidence of adverse events (AEs), and complete stone removal rate. RESULTS: Of the 54 patients enrolled, 23 underwent the EUS-AG procedure and 31 DB-ERC. The technical success rates of EUS-AG and DB-ERC were 87.0% and 64.5%, respectively (P = 0.11). The procedure time was significantly shorter in the EUS-AG group than in the DB-ERC group (51.9 ± 15.4 vs 72.6 ± 32.2 min; P = 0.01), and the early AE rates were 26.1% and 12.9%, respectively (P = 0.71). The complete stone removal rates in patients who underwent previous stone removal were 94.1% in the EUS-AG group and 85.7% in the DB-ERC group (P = 0.61). CONCLUSION: The EUS-AG afforded technical success and complete stone removal rates comparable with those of DB-ERC, but the former procedure was shorter. The AE rate was acceptable.

Endoscopic Ultrasonography Findings of Early and Suspected Early Chronic Pancreatitis.

Chronic pancreatitis (CP) is associated with a risk of pancreatic cancer and is characterized by irreversible morphological changes, fibrosis, calcification, and exocrine and endocrine insufficiency. CP is a progressive disease with a poor prognosis and is typically diagnosed at an advanced stage. The Japan Pancreas Society proposed criteria for early CP in 2009, and their usefulness has been reported. Recently, a mechanism definition was proposed by the International Consensus Guidelines and early CP was defined as a disease state that is not based on disease duration. CP is diagnosed by computed tomography, magnetic resonance imaging, and endoscopic cholangiopancreatography, which can detect calcification and dilation of the pancreatic ducts; however, detecting early CP with these modalities is difficult because subtle changes in early CP occur before established CP or end-stage CP. Endoscopic ultrasonography (EUS) is useful in the diagnosis of early CP because it allows high-resolution, close-up observation of the pancreas. In addition to imaging findings, EUS with elastography enables measurement of the stiffness of the pancreas, an objective diagnostic measure. Understanding the EUS findings of early CP is important because a histological diagnosis is problematic, and other modalities are not capable of detecting subtle changes in early CP.

Clinical Evaluation of a Newly Developed Guidewire for Pancreatobiliary Endoscopy.

BACKGROUND: The guidewire (GW) plays an important role in pancreatobiliary endoscopy. GW quality is a critical factor in the effectiveness and efficiency of pancreatobiliary endoscopy. In this study, we evaluate a new 0.025 inch multipurpose endoscopic GW: the M-Through. METHODS: Our study was a multicenter retrospective analysis. We enrolled patients who underwent endoscopic procedures using the M-Through between May 2018 and April 2020. Patients receiving the following endoscopic treatments were enrolled: common bile duct (CBD) stone extraction, endoscopic drainage for distal and hilar malignant biliary obstruction (MBO), and endoscopic drainage for acute cholecystitis. For each procedure, we examined the rate of success without GW exchange. RESULTS: A total of 170 patients (80 with CBD stones, 60 with MBO, and 30 with cholecystitis) were enrolled. The rate of completion without GW exchange was 100% for CBD stone extraction, 83.3% for endoscopic drainage for MBO, and 43.3% for endoscopic drainage for cholecystitis. In unsuccessful cholecystitis cases with the original GW manipulator, 1 of 8 cases succeeded in the manipulator exchange. Including 6 cases who changed GW after the manipulator exchange, 11 of 16 cases succeeded in changing GW. There was significant difference in the success rate between the manipulator exchange and GW exchange (p = 0.03). The insertion of devices and stent placement after biliary cannulation (regardless of type) were almost completed with M-through. We observed no intraoperative GW-related adverse events such as perforation and bleeding due to manipulation. CONCLUSION: The 0.025 inch M-Through can be used for endoscopic retrograde cholangiopancreatography-related procedures efficiently and safely. Our study found high rates of success without GW exchange in all procedures except for endoscopic drainage for cholecystitis. This GW is considered (1) excellent for supportability of device insertion to remove CBD stones; (2) good for seeking the biliary malignant stricture but sometimes need the help of a hydrophilic GW; (3) suboptimal for gallbladder drainage that require a high level of seeking ability.

Best Procedure for the Management of Common Bile Duct Stones via the Papilla: Literature Review and Analysis of Procedural Efficacy and Safety.

BACKGROUND: Endoscopic management of common bile duct stones (CBDS) is standard; however, various techniques are performed via the papilla, and the best procedure in terms of both efficacy and safety has not been determined. METHODS: Endoscopic procedures were classified into five categories according to endoscopic sphincterotomy (EST) and balloon dilation (BD): (1) EST, (2) endoscopic papillary BD (≤10 mm) (EPBD), (3) EST followed by BD (≤10 mm) (ESBD), (4) endoscopic papillary large BD (≥12 mm) (EPLBD), and (5) EST followed by large BD (≥12 mm) (ESLBD). We performed a literature review of prospective and retrospective studies to compare efficacy and adverse events (AEs). Each procedure was associated with different efficacy and AE profiles. RESULTS: In total, 19 prospective and seven retrospective studies with a total of 3930 patients were included in this study. For EST, the complete stone removal rate at the first session, rate of mechanical lithotripsy (ML), and rate of overall AEs in EST were superior to EPBD, but a higher rate of bleeding was found for EST. Based on one retrospective study, complete stone removal rate at the first session, rate of ML, and rate of overall AEs were superior for ESBD vs. EST, and the rate of bleeding for the former was also lower. Complete stone removal rate at the first session and rate of ML for ESLBD were superior to those for EST, with no significant difference in rate of AEs. For EST vs. EPLBD, complete stone removal rate at the first session and rate of ML were superior for the latter. For EPLBD vs. ESLBD, the efficacy and safety were similar. CONCLUSIONS: ESBD is considered the best procedure for the management of small CBDS, but strong evidence is lacking. For large CBDS, both ESLBD and EPLBD are similar.

Evaluation of the safety and efficacy of minimal endoscopic sphincterotomy followed by papillary balloon dilation for the removal of common bile duct stones.

BACKGROUND/AIM: A sufficiently open papilla is needed to remove common bile duct stones (CBDS) but endoscopic sphincterotomy (EST) requires a high level of skill and is difficult with endoscopic papillary balloon dilation (EPBD). The main adverse event of EST is bleeding and perforation and that of EPBD is post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis. To reduce these adverse events we employed minimal EST followed by papillary dilation (ESBD), and retrospectively evaluated its efficacy and safety compared with EST. PATIENTS AND METHODS: CBDS patients who underwent EST (n = 114) or ESBD (n = 321) at Juntendo University Hospital from January 2009 to December 2018 were consecutively enrolled, retrospectively. The exclusion criteria were large-balloon dilation (≥ 12 mm), large CBDS (>12 mm), and previous EST/EPBD. We compared the overall stone removal rate, incidence of adverse event, procedure time, number of ERCP procedures, and rate of mechanical lithotripsy (ML) between the two groups. RESULTS: Complete stone removal was successful in both ESBD and EST group. However, the rate of multiple ERCP sessions was significantly lower (35.1% vs. 12.8%, P < 0.001), procedure time was shorter (31.6 vs. 25.8 min, P = 0.01), and rate of ML was lower (16.7% vs. 7.8%, P = 0.01) in ESBD group. Bleeding was significantly more frequent in the EST group (9.6% vs. 1.2%, P < 0.001), particularly acute bleeding (7.9% vs. 0.9%, P < 0.001). CONCLUSIONS: ESBD is more efficient and safer in the management of CBD stones than EST. A prospective randomized study comparing ESBD with EST is needed to establish this combination technique.

Role of Peroral Cholangioscopy in the Diagnosis of Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by idiopathic biliary stricture followed by progressive cholestasis and fibrosis. When diagnosing PSC, its differentiation from other types of sclerosing cholangitis and cholangiocarcinoma is necessary. The cholangioscopic findings of PSC have not been investigated sufficiently. PSC and IgG4-related sclerosing cholangitis are difficult to distinguish by peroral cholangioscopy (POCS), but POCS is useful for excluding cholangiocarcinoma. POCS findings vary according to the condition and stage of disease. In the active phase, findings such as mucosal erythema, ulceration, fibrinous white exudate, and an irregular surface are observed and may reflect strong inflammation in the biliary epithelium. On the other hand, findings such as scarring, pseudodiverticula, and bile duct stenosis appear in the chronic phase and may reflect fibrosis and stenosis resulting from repeated inflammation. Observation of inside the bile duct by POCS might confirm the current PSC activity. Because POCS offers not only information regarding the diagnosis of PSC and PSC-associated cholangiocarcinoma but also the current statuses of biliary inflammation and stenosis, POCS could significantly contribute to the diagnosis and treatment of PSC once the characteristic findings of PSC are confirmed by future studies.

Elevation of Neutrophil-to-Lymphocyte Ratio before First-Line Chemotherapy Predicts a Poor Prognosis for Second-Line Chemotherapy in Gastric Cancer.

OBJECTIVES: The neutrophil-to-lymphocyte ratio (NLR) has been proposed as an indicator of cancer-related inflammation. The aim of our study was to examine the prognostic value of the NLR for patients with advanced gastric cancer receiving second-line chemotherapy. METHODS: The association of overall survival (OS) in second-line chemotherapy and the clinicopathological findings including NLR were analyzed retrospectively. The selection criteria were patients who received second-line chemotherapy between January 2010 and June 2015, had histologically confirmed gastric adenocarcinoma, and were followed up until death or for 180 days or longer. RESULTS: Eighty-six patients met the selection criteria. Multivariate analysis revealed that performance status 2, hemoglobin < 10 g/dL, and NLR before first-line chemotherapy ≥3 were adverse predictive markers. NLR before second-line chemotherapy was not associated with OS. A prognostic model was constructed dividing patients into three groups according to the number of adverse predictive factors: good (no factor), intermediate (one factor), and poor (more than two factors). The median OS for the good, intermediate, and poor groups was 14.3, 7.2, and 4.4 months, respectively (p < 0.001). CONCLUSIONS: Patients with advanced gastric cancer with performance status 2, hemoglobin < 10 g/dL, and NLR before first-line chemotherapy ≥3 are not likely to benefit from second-line chemotherapy.

X-Ray snapshots of a pyridoxal enzyme: a catalytic mechanism involving concerted [1,5]-hydrogen sigmatropy in methionine γ-lyase.

Pyridoxal 5'-phosphate (PLP)-enzymes are essentially involved in amino acid and amine metabolism of a wide variety of organisms. Despite their extensive biochemical studies, there are little evidence and structural data to comprehensively elaborate the catalytic mechanism. We obtained X-ray snapshots of l-methionine γ-lyase from Entamoeba histolytica (EhMGL), a PLP-enzyme catalyzing the γ-elimination reaction of methionine. Here, we suggest a catalytic mechanism of EhMGL by using the X-ray snapshots covering all stages of this multistep catalysis reaction. Initial formation of a Michaelis complex is followed by the migration of double bond from the C4'=Nα-Cα moiety in an intermediate PLP-methionine imine to C4'-Nα=Cα in pyridoxamine 5'-phosphate (PMP)-α,ß-dehydromethionine imine without intervention of a putative quinonoid intermediate. The enzyme can facilitate the subsequent γ-elimination of methanethiol by the possible general acid-base catalysis of Tyr108 for the E1cB mechanism, enabling to form the ene-imine C4'-Nα=Cα-Cß=Cγ structure with the s-cis conformation, which is prerequisite for the non-enzymatic symmetry-allowed suprafacial [1,5]-hydrogen shift to complete the catalytic cycle by releasing α-ketobutyrate. The mechanism based on the X-ray snapshots is consistent with the reactivity of MGL toward methionine analogues. The generality of such a mechanism involving non-enzymatic concerted reaction in other PLP enzymes is discussed.

Kinetic characterization of methionine gamma-lyases from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis.

Methionine gamma-lyase (MGL) (EC 4.4.1.11), which is present in certain lineages of bacteria, plants, and protozoa but missing in mammals, catalyzes the single-step degradation of sulfur-containing amino acids (SAAs) to alpha-keto acids, ammonia, and thiol compounds. In contrast to other organisms possessing MGL, anaerobic parasitic protists, namely Entamoeba histolytica and Trichomonas vaginalis, harbor a pair of MGL isozymes. The enteric protozoon En. histolytica shows various unique aspects in its metabolism, particularly degradation of SAAs. Trifluoromethionine (TFM), a halogenated analog of Met, has been exploited as a therapeutic agent against cancer as well as against infections by protozoan organisms and periodontal bacteria. However, its mechanism of action remains poorly understood. In addition, the physiological significance of the presence of two MGL isozymes in these protists remains unclear. In this study, we compared kinetic parameters of the wild-type and mutants, engineered by site-directed mutagenesis, of the two MGL isotypes from En. histolytica (EhMGL1 and EhMGL2) for various potential substrates and TFM. Intracellular concentrations of l-Met and l-Cys suggested that these SAAs are predominantly metabolized by EhMGL1, not by EhMGL2. It is unlikely that O-acetyl-l-serine is decomposed by EhMGLs, given the kinetic parameters of cysteine synthase reported previously. Comparison of the wild-type and mutants revealed that the contributions of several amino acids implicated in catalysis differ between the two isozymes, and that the degradation of TFM is less sensitive to alterations of these residues than is the degradation of physiological substrates. These results support the use of TFM to target MGL.

Expression, purification and crystallization of L-methionine gamma-lyase 2 from Entamoeba histolytica.

L-Methionine gamma-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 A. The crystal diffracted to a resolution of 2.0 A. The presence of a tetramer in the asymmetric unit (4 x 43.1 kDa) gives a Matthews coefficient of 2.2 A(3) Da(-1). The structure was solved by the molecular-replacement method and structure refinement is now in progress.