Metagenomic characterization of the equine endometrial microbiome during anestrus.

The equine uterus is highly interrogated during estrus prior to breeding and establishing pregnancy. Many studies in mares have been performed during estrus under the influence of high estrogen concentrations, including the equine estrual microbiome. To date, it is unknown how the uterine microbiome of the mare is influenced by cyclicity; while, the equine vaginal microbiome is stable throughout the estrous cycle. We hypothesized that differences would exist between the equine endometrial microbiome of mares in estrus and anestrus. The aim of this study was two-fold: to characterize the resident endometrial microbiome of healthy mares during anestrus and to compare this with estrus. Double-guarded endometrial swabs were taken from healthy mares during estrus (n = 16) and in the following non-breeding season during anestrus (n = 8). Microbial population was identified using 16S rRNA sequencing. Our results suggest that the equine uterine microbiome in estrus has a low diversity and low richness, while during anestrus, a higher diversity and higher richness were seen compared to estrus. Despite this difference, both the estrus and anestrus endometrial microbiome were dominated by Proteobacteria, Firmicutes, and Bacteroidota. The composition of the microbial community between anestrus and estrus was significantly different. This may be explained by the difference in the composition of the endometrial immune milieu based on the stage of the cycle. Further research investigating the function of the equine endometrial microbiome and dynamics changes within the uterine environment is required.

Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus.

Bacterial endometritis is among the most common causes of subfertility in mares. It has a major economic impact on the equine breeding industry. The sensitivity of detecting uterine microbes using culture-based methods, irrespective of the sample collection method, double-guarded endometrial swab, endometrial biopsy, or uterine low-volume lavage (LVL), is low. Therefore, equine bacterial endometritis often goes undiagnosed. Sixteen individual mares were enrolled, and an endometrial sample was obtained using each method from all mares. After trimming, quality control and decontamination, 3824 amplicon sequence variants were detected in the dataset. We found using 16S rRNA sequencing that the equine uterus harbors a distinct resident microbiome during estrus. All three sampling methods used yielded similar results in composition as well as relative abundance at phyla (Proteobacteria, Firmicutes, and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) levels. A significant difference was found in alpha diversity (Chao1) between LVL and endometrial biopsy, suggesting that LVL is superior at detecting the low-abundant (rare) taxa. These new data could pave the way for innovative treatment methods for endometrial disease and subfertility in mares. This, in turn, could lead to more judicious antimicrobial use in the equine breeding industry.

Flatulence After Anti-reflux Treatment (FAART) Study.

BACKGROUND: Flatulence is known to be a common side effect of laparoscopic fundoplication, yet the true incidence is unclear and its impact on patients' quality of life not well understood. This study aimed to assess the long-term incidence of flatulence, and its effect on quality of life, following fundoplication. METHODS: All patients who underwent primary laparoscopic fundoplication between 1999 and 2009 were identified from a prospectively maintained institutional database. A cross-sectional analysis of post-operative gastrointestinal symptoms and quality of life was performed using a symptom-specific questionnaire. Statistical analysis of outcomes stratified by sex and type of fundoplication was performed. RESULTS: 462 eligible patients were identified from the database, with follow-up obtained in 265 (57%). Median age at surgery was 53 (22-78) years. 137 patients (52%) were female. 138 (52%) underwent a 360° fundoplication, the remainder a partial fundoplication. At median follow-up of 11 (8-15) years, excessive flatulence was reported by 85%. Only 12% reported an adverse impact on social life, and 11% an adverse impact on quality of life. Flatulence was worse following a total than partial fundoplication, women reported more gas-related symptoms than men, yet neither sex nor wrap type had a significant impact on social life or quality of life. CONCLUSIONS: The majority of patients report excessive flatulence at long-term follow-up after anti-reflux surgery, yet the impact on social life and quality life was small. There was no evidence to support tailoring of wrap type by sex to avoid gas-related symptoms. The authors advocate that all patients understand the inevitable side effects of fundoplication to help manage expectations from surgery.

Development of a risk prediction model for Barrett's esophagus in an Australian population.

Esophageal adenocarcinoma has poor 5-year survival rates. Increased survival might be achieved with earlier treatment, but requires earlier identification of the precursor, Barrett's esophagus. Population screening is not cost effective, this may be improved by targeted screening directed at individuals more likely to have Barrett's esophagus. To develop a risk prediction tool for Barrett's esophagus, this study compared individuals with Barrett's esophagus against population controls. Participants completed a questionnaire comprising 35 questions addressing medical history, symptom history, lifestyle factors, anthropomorphic measures, and demographic details. Statistical analysis addressed differences between cases and controls, and entailed initial variable selection, checking of model assumptions, and establishing calibration and discrimination. The area under the curve (AUC) was used to assess overall accuracy. One hundred and twenty individuals with Barrett's esophagus and 235 population controls completed the questionnaire. Significant differences were identified for age, gender, reflux history, family reflux history, history of hypertension, alcoholic drinks per week, and body mass index. These were used to develop a risk prediction model. The AUC was 0.82 (95% CI 0.78-0.87). Good calibration between predicted and observed risk was noted (Hosmer-Lemeshow test P = 0.67). At the point minimizing false positives and false negatives, the model achieved a sensitivity of 84.96% and a specificity of 66%. A well-calibrated risk prediction model with good discrimination has been developed to identify patients with Barrett's esophagus. The model needs to be externally validated before consideration for clinical practice.

Risk factors for delayed gastric emptying following laparoscopic repair of very large hiatus hernias.

BACKGROUND: Delayed gastric emptying can complicate surgery for hiatus hernia. The aim of this study was to quantify its incidence following laparoscopic repair of very large hiatus hernias, identify key risk factors for its occurrence and determine its impact on clinical outcomes. METHODS: Data collected from a randomized trial of patients who underwent laparoscopic mesh versus sutured repair of very large hiatus hernias (more than 50 per cent of stomach in chest) were analysed retrospectively. Delayed gastric emptying was defined as endoscopic evidence of solid food in the stomach after fasting for 6 h at 6 months after surgery. RESULTS: Delayed gastric emptying occurred in 19 of 102 patients (18·6 per cent). In univariable analysis, type 2 paraoesophageal hernia (relative risk (RR) 3·15, 95 per cent c.i. 1·41 to 7·06), concurrent anterior and posterior hiatal repair (RR 2·66, 1·14 to 6·18), hernia sac excision (RR 4·85, 1·65 to 14·24), 270°/360° fundoplication (RR 3·64, 1·72 to 7·68), division of short gastric vessels (RR 6·82, 2·12 to 21·90) and revisional surgery (RR 3·69, 1·73 to 7·87) correlated with delayed gastric emptying. In multivariable analysis, division of short gastric vessels (RR 6·27, 1·85 to 21·26) and revisional surgery (RR 6·19, 1·32 to 28·96) were independently associated with delayed gastric emptying. Delayed gastric emptying correlated with adverse gastrointestinal symptomatology, including higher rates of bloating, nausea, vomiting and anorexia, as well as reduced patient satisfaction with the operation and recovery. CONCLUSION: Delayed gastric emptying following large hiatus hernia repair is common and associated with adverse symptoms and reduced patient satisfaction. Division of short gastric vessels and revisional surgery were independently associated with its occurrence.

Venous thromboembolism in patients with esophageal or gastric cancer undergoing neoadjuvant chemotherapy.

There is a well-established link between cancer and venous thromboembolism (VTE), and patients receiving chemotherapy for esophageal or gastric cancer appear at high risk of developing VTE. The incidence of VTE in the neoadjuvant setting in these patients is poorly understood, as is the role for thromboprophylaxis during neoadjuvant chemotherapy. A PubMed search was conducted using a combination of terms including; esophageal & gastric cancer, deep venous thrombosis (DVT), VTE, neoadjuvant, chemotherapy and chemoradiotherapy. One hundred and fifty-four articles were retrieved and a narrative review was conducted. For patients with esophageal and gastric cancer the incidence of VTE ranged from 4 to 19%. Gastric cancer (Odds Ratio [OR] 6.38, [95% CI: 1.96-20.80]) and Stage III/IV disease, (OR 5.16 [95% CI: 1.29-20.73]) were identified as risk factors for developing VTE. Neoadjuvant chemotherapy was identified as an independent risk factor for developing VTE. Symptomatic and asymptomatic VTE have a similar effect on mortality. Median overall survival for asymptomatic VTE was 13.9 months (95% CI: 5.0-∞) versus 12.8 months (95% CI: 4.7-30.3) if the VTE was symptomatic. Neoadjuvant chemotherapy is a significant risk factor for VTE in patients with esophageal and gastric cancer. Intervention to minimize the risk using pharmacological and mechanical thromboprophylaxis should be considered, and this should start in the neoadjuvant period.

Evaluation of outcome after cardiomyotomy for achalasia using the Chicago classification.

BACKGROUND: Achalasia can be subdivided into manometric subtypes according to the Chicago classification. These subtypes are proposed to predict outcome after treatment. This hypothesis was tested using a database of patients who underwent laparoscopic Heller's cardiomyotomy with anterior fundoplication. METHODS: All patients who underwent Heller's cardiomyotomy for achalasia between June 1993 and March 2015 were identified from an institutional database. Manometry tracings were retrieved and re-reported according the Chicago classification. Outcome was assessed by a postal questionnaire, and designated a success if the modified Eckardt score was 3 or less, and the patient had not undergone subsequent surgery or pneumatic dilatation. Difference in outcome after cardiomyotomy was analysed with a mixed-effects logistic regression model. RESULTS: Sixty, 111 and 24 patients had type I, II and II achalasia respectively. Patients with type III achalasia were more likely to be older than those with type I or II (mean age 63 versus 50 and 49 years respectively; P = 0·001). Some 176 of 195 patients returned questionnaires after surgery. Type III achalasia was less likely to have a successful outcome than type II (odds ratio (OR) 0·38, 95 per cent c.i. 0·15 to 0·94; P = 0·035). There was no significant difference in outcome between types I and II achalasia (II versus I: OR 0·87, 0·47 to 1·60; P 0·663). The success rate at 3-year follow-up was 69 per cent (22 of 32) for type I, 66 per cent (33 of 50) for type II and 31 per cent (4 of 13) for type III. CONCLUSION: Type III achalasia is a predictor of poor outcome after cardiomyotomy. There was no difference in outcome between types I and II achalasia.

Novel handheld magnetometer probe based on magnetic tunnelling junction sensors for intraoperative sentinel lymph node identification.

Using magnetic tunnelling junction sensors, a novel magnetometer probe for the identification of the sentinel lymph node using magnetic tracers was developed. Probe performance was characterised in vitro and validated in a preclinical swine model. Compared to conventional gamma probes, the magnetometer probe showed excellent spatial resolution of 4.0 mm, and the potential to detect as few as 5 µg of magnetic tracer. Due to the high sensitivity of the magnetometer, all first-tier nodes were identified in the preclinical experiments, and there were no instances of false positive or false negative detection. Furthermore, these preliminary data encourage the application of the magnetometer probe for use in more complex lymphatic environments, such as in gastrointestinal cancers, where the sentinel node is often in close proximity to other non-sentinel nodes, and high spatial resolution detection is required.

Tissue invasion and metastasis: Molecular, biological and clinical perspectives.

Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), ß-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-ß), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks.

Pre-therapy mRNA expression of TNF is associated with regimen-related gastrointestinal toxicity in patients with esophageal cancer: a pilot study.

PURPOSE: Esophageal cancer has a high mortality rate, and its multimodality treatment is often associated with significant rates of severe toxicity. Effort is needed to uncover ways to maximize effectiveness of therapy through identification of predictive markers of response and toxicity. As such, the aim of this study was to identify genes predictive of chemoradiotherapy-induced gastrointestinal toxicity using an immune pathway-targeted approach. METHODS: Adults with esophageal cancer treated with chemotherapy consisting of 5-fluorouracil and cisplatin and 45-50 Gy radiation were recruited to the study. Pre-therapy-collected whole blood was analyzed for relative expression of immune genes using real-time polymerase chain reaction (RT-PCR). Gene expression was compared between patients who experienced severe regimen-related gastrointestinal toxicity vs. those experiencing mild to moderate toxicity. RESULTS: Blood from 31 patients were analyzed by RT-PCR. Out of 84 immune genes investigated, TNF was significantly elevated (2.05-fold, p = 0.025) in the toxic group (n = 12) compared to the non-toxic group (n = 19). Nausea and vomiting was the most commonly documented severe toxicity. No associations between toxicity and response, age, sex, histology, or treatment were evident. CONCLUSIONS: This study supports evidence of TNF as a predictive biomarker in regimen-related gastrointestinal toxicity. Confirming these findings in a larger cohort is warranted.

Molecular physiognomies and applications of adipose-derived stem cells.

Adipose-derived stromal/stem cells (ASC) are multipotent with abilities to differentiate into multiple lineages including connective tissue and neural cells. Despite unlimited opportunity and needs for human and veterinary regenerative medicine, applications of adipose-derived stromal/stem cells are at present very limited. Furthermore, the fundamental biological factors regulating stemness in ASC and their stable differentiation into other tissue cells are not fully understood. The objective of this review was to provide an update on the current knowledge of the nature and isolation, molecular and epigenetic determinants of the potency, and applications of adipose-derived stromal/stem cells, as well as challenges and future directions. The first quarter of the review focuses on the nature of ASC, namely their definition, origin, isolation and sorting methods and multilineage differentiation potential, often with a comparison to mesenchymal stem cells of bone marrow. Due to the indisputable role of epigenetic regulation on cell identities, epigenetic modifications (DNA methylation, chromatin remodeling and microRNAs) are described broadly in stem cells but with a focus on ASC. The final sections provide insights into the current and potential applications of ASC in human and veterinary regenerative medicine.

Twenty years of experience with laparoscopic antireflux surgery.

BACKGROUND: There are few reports of large patient cohorts with long-term follow-up after laparoscopic antireflux surgery. This study was undertaken to evaluate changes in surgical practice and outcomes for laparoscopic antireflux surgery over a 20-year period. METHODS: A standardized questionnaire, prospectively applied annually, was used to determine outcome for all patients undergoing laparoscopic fundoplication in two centres since commencing this procedure in 1991. Visual analogue scales ranging from 0 to 10 were used to assess symptoms of heartburn, dysphagia and satisfaction with overall outcome. Data were analysed to determine outcome across 20 years. RESULTS: From 1991 to 2010, 2261 consecutive patients underwent laparoscopic fundoplication at the authors' institutions. Follow-up ranged from 1 to 19 (mean 7.6) years. Conversion to open surgery occurred in 73 operations (3.2 per cent). Revisional surgery was performed in 216 patients (9.6 per cent), within 12 months of the original operation in 116. There was a shift from Nissen to partial fundoplication across 20 years, and a recent decline in operations for reflux, offset by an increase in surgery for large hiatus hernia. Dysphagia and satisfaction scores were stable, and heartburn scores rose slightly across 15 years of follow-up. Heartburn scores were slightly higher and reoperation for reflux was more common after anterior partial fundoplication (P = 0.005), whereas dysphagia scores were lower and reoperation for dysphagia was less common (P < 0.001). At 10 years, satisfaction with outcome was similar for all fundoplication types. CONCLUSION: Laparoscopic Nissen and partial fundoplications proved to be durable and achieved good long-term outcomes. At earlier follow-up, dysphagia was less common but reflux more common after anterior partial fundoplication, although differences had largely disappeared by 10 years.

Post-fundoplication contrast studies: is there room for improvement?

OBJECTIVE: Since the mid-1990s, laparoscopic fundoplication for gastro-oesophageal reflux disease has become the surgical procedure of choice. Several surgical groups perform routine post-operative contrast studies to exclude any (asymptomatic) anatomical abnormality and to expedite discharge from hospital. The purpose of this study was to determine the accuracy and interobserver reliability for surgeons and radiologists in contrast study interpretation. METHODS: 11 surgeons and 13 radiologists (all blinded to outcome) retrospectively reviewed the contrast studies of 20 patients who had undergone a laparoscopic fundoplication. Each observer reported on fundal wrap position, leak or extravasation of contrast and contrast hold-up at the gastro-oesophageal junction (on a scale of 0-4). A κ coefficient was used to evaluate interobserver reliability. RESULTS: Surgeons were more accurate than radiologists in identifying normal studies (specificity = 91.6% vs 78.9%), whereas both groups had similar accuracy in identifying abnormal studies (sensitivity = 82.3% vs 85.2%). There was higher agreement amongst surgeons than amongst radiologists when determining wrap position (κ = 0.65 vs 0.54). Both groups had low agreement when classifying a wrap migration as partial or total (κ = 0.33 vs 0.06). Radiologists were more likely to interpret the position of the wrap as abnormal (relative risk = 1.25) while surgeons reported a greater degree of hold-up of contrast at the gastro-oesophageal junction (mean score = 1.17 vs 0.86). CONCLUSION: Radiologists would benefit from more information about the technical details of laparoscopic anti-reflux surgery. Standardised protocols for performing post-fundoplication contrast studies are needed.

Impact of ineffective oesophageal motility and wrap type on dysphagia after laparoscopic fundoplication.

BACKGROUND: Laparoscopic 360° fundoplication is the most common operation for gastro-oesophageal reflux disease, but is associated with postoperative dysphagia in some patients. Patients with ineffective oesophageal motility may have a higher risk of developing postoperative dysphagia, but this remains unclear. METHODS: From 1991 to 2010, 2040 patients underwent primary laparoscopic fundoplication for gastro-oesophageal reflux disease and met the study inclusion criteria; 343 had a 90°, 498 a 180° and 1199 a 360° fundoplication. Primary peristalsis and distal contraction amplitude during oesophageal manometry were determined for 1354 patients. Postoperative dysphagia scores (range 0-45) were recorded at 3 and 12 months, then annually. Oesophageal dilatations and/or reoperations for dysphagia were recorded. RESULTS: Preoperative oesophageal motility did not influence postoperative dysphagia scores, the need for dilatation and/or reoperation up to 6 years. Three-month dysphagia scores were lower after 90° and 180° compared with 360° fundoplication (mean(s.e.m.) 8·0(0·6) and 9·8(0·5) respectively versus 11·9(0·4); P < 0·001 and P = 0·003), but these differences diminished after 6 years of follow-up. The incidence of dilatation and reoperation for dysphagia was lower after 90° (2·6 and 0·6 per cent respectively) and 180° (4·4 and 1·0 per cent) fundoplications than with a 360° wrap (9·8 and 6·8 per cent; both P < 0·001 versus 90° and 180° groups). CONCLUSION: Tailoring the degree of fundoplication according to preoperative oesophageal motility by standard manometric parameters has no long-term impact on postoperative dysphagia. There is, however, a proportionate increase in short-term dysphagia scores with increasing degree of wrap, and a corresponding proportionate increase in dilatations and reoperations for dysphagia. These differences in dysphagia scores diminish with time.

Diffuse esophageal leiomyomatosis.

Progressive dysphagia of unknown etiology may still provide a diagnostic challenge despite an increase in the number and quality of investigations available. We describe a 64-year-old man who presented with progressive dysphagia and weight loss. Following a number of investigations, a diagnosis of diffuse esophageal leiomyomatosis was made and the patient was treated appropriately.

Long-term outcomes of revisional surgery following laparoscopic fundoplication.

BACKGROUND: A small proportion of patients who have laparoscopic antireflux procedures require revisional surgery. This study investigated long-term clinical outcomes. METHODS: Patients requiring late revisional surgery following laparoscopic fundoplication for gastro-oesophageal reflux were identified from a prospective database. Long-term outcomes were determined using a questionnaire evaluating symptom scores for heartburn, dysphagia and satisfaction. RESULTS: The database search found 109 patients, including 98 (5.6 per cent) of 1751 patients who had primary surgery in the authors' unit. Indications for surgical revision were dysphagia (52 patients), recurrent reflux (36), mechanical symptoms related to paraoesophageal herniation (16) and atypical symptoms (five). The median time to revision was 26 months. Outcome data were available for 104 patients (median follow-up 66 months) and satisfaction data for 102, 88 of whom were highly satisfied (62.7 per cent) or satisfied (23.5 per cent) with the outcome. Patients who had revision for dysphagia had a higher incidence of poorly controlled heartburn (20 versus 2 per cent; P = 0.004), troublesome dysphagia (16 versus 6 per cent; P = 0.118) and a lower satisfaction score (P = 0.023) than those with recurrent reflux or paraoesophageal herniation. CONCLUSION: Revisional surgery following laparoscopic fundoplication can produce good long-term results, but revision for dysphagia has less satisfactory outcomes.

Detection of lymph node metastases in oesophageal cancer.

BACKGROUND: The importance of lymph node status in oesophageal cancer cannot be disputed. It is therefore surprising that no standardization exists in either terminology or methodology in lymph node analysis. METHODS: All online databases were searched to identify articles published from 1970 onwards. This was supplemented by hand searching references of retrieved articles. RESULTS AND CONCLUSION: Without accurate identification of lymph node metastases, patients cannot be staged properly, nor can best practice for the treatment of oesophageal cancer be determined. This review outlines the problem and proposes recommendations for standardization in terminology and methodology for the detection of lymph node metastases in oesophageal cancer.

Risk prediction scores for postoperative mortality after esophagectomy: validation of different models.

BACKGROUND: Different prediction models for operative mortality after esophagectomy have been developed. The aim of this study is to independently validate prediction models from Philadelphia, Rotterdam, Munich, and the ASA. METHODS: The scores were validated using logistic regression models in two cohorts of patients undergoing esophagectomy for cancer from Switzerland (n = 170) and Australia (n = 176). RESULTS: All scores except ASA were significantly higher in the Australian cohort. There was no significant difference in 30-day mortality or in-hospital death between groups. The Philadelphia and Rotterdam scores had a significant predictive value for 30-day mortality (p = 0.001) and in-hospital death (p = 0.003) in the pooled cohort, but only the Philadelphia score had a significant prediction value for 30-day mortality in both cohorts. Neither score showed any predictive value for in-hospital death in Australians but were highly significant in the Swiss cohort. ASA showed only a significant predictive value for 30-day mortality in the Swiss. For in-hospital death, ASA was a significant predictor in the pooled and Swiss cohorts. The Munich score did not have any significant predictive value whatsoever. CONCLUSION: None of the scores can be applied generally. A better overall predictive score or specific prediction scores for each country should be developed.

Steady-state kinetic characterization of substrates and metal-ion specificities of the full-length and N-terminally truncated recombinant human methionine aminopeptidases (type 2).

The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested.