Pulmonary oedema in the course of severe maternal outcome in South Africa: A cohort study combined with clinical audit.

OBJECTIVES: To describe the incidence and outcomes of pulmonary oedema in women with severe maternal outcome during childbirth and identify possible modifiable factors through audit. METHODS: All women with severe maternal outcome (maternal deaths or near misses) who were referred to Tygerberg referral hospital from health facilities in Metro East district, South Africa, during 2014-2015 were included. Women with severe maternal outcome and pulmonary oedema during pregnancy or childbirth were evaluated using three types of critical incident audit: criterion-based case review by one consultant gynaecologist, monodisciplinary critical incident audit by a team of gynaecologists, multidisciplinary audit with expert review from anaesthesiologists and cardiologists. RESULTS: Of 32,161 pregnant women who gave birth in the study period, 399 (1.2%) women had severe maternal outcome and 72/399 (18.1%) had pulmonary oedema with a case fatality rate of 5.6% (4/72). Critical incident audit demonstrated that pre-eclampsia/HELLP-syndrome and chronic hypertension were the main conditions underlying pulmonary oedema (44/72, 61.1%). Administration of volumes of intravenous fluids in already sick women, undiagnosed underlying cardiac illness, administration of magnesium sulphate as part of pre-eclampsia management and oxytocin for augmentation of labour were identified as possible contributors to the pathophysiology of pulmonary oedema. Women-related factors (improved antenatal care attendance) and health care-related factors (earlier diagnosis and management) would potentially have improved maternal outcome. CONCLUSIONS: Although pulmonary oedema in pregnancy is rare, among women with severe maternal outcome a considerable proportion had pulmonary oedema (18.1%). Audit identified options for prevention of pulmonary oedema and improved outcome. These included early detection and management of preeclampsia with close monitoring of fluid intake and cardiac evaluation in case of suspected pulmonary oedema. Therefore, a multidisciplinary clinical approach is recommended.

Stalled at the intersection: insurance status and disparities in post-mastectomy breast reconstruction.

PURPOSE: Post-mastectomy breast reconstruction (PMBR) is an important component of breast cancer treatment, but disparities relative to insurance status persist despite legislation targeting the issue. We aimed to study this relationship in a large health system combining a safety-net hospital and a private academic center. METHODS: Data were collected on all patients who underwent mastectomy for breast cancer from 2011 to 2019 in a private academic center and an adjacent public safety-net hospital served by the same surgical teams. Multivariable logistic regression was used to assess the effect of insurance status on PMBR, controlling for covariates that included socioeconomic, demographic, and clinical factors. RESULTS: Of 1554 patients undergoing mastectomy for breast cancer, 753 (48.5%) underwent PMBR, of which 592 (79.9%) were privately insured, 50 (6.7%) Medicare, 68 (9.2%) Medicaid, and 31 (4.2%) uninsured. Multivariable logistic regression showed a significantly higher likelihood of not undergoing PMBR for uninsured (OR 6.0, 95% CI 3.7-9.8; p < 0.0001), Medicare (OR 1.9, (95% CI 1.2-3.0; p = 0.006), and Medicaid (OR 1.5, 95% CI 1.0-2.3; p = 0.04) patients compared with privately insured patients. Age, stage, race and ethnicity, and hospital type confounded this relationship. CONCLUSION: Patients without health insurance have dramatically reduced access to PMBR compared to those with private insurance. Expanding access to this important procedure is essential to achieve greater health equity for breast cancer patients.

Recommendations on dose level selection for repeat dose toxicity studies.

Prior to registering and marketing any new pharmaceutical, (agro)chemical or food ingredient product manufacturers must, by law, generate data to ensure human safety. Safety testing requirements vary depending on sector, but generally repeat-dose testing in animals form the basis for human health risk assessments. Dose level selection is an important consideration when designing such studies, to ensure that exposure levels that lead to relevant hazards are identified. Advice on dose level selection is provided in test guidelines and allied guidance documents, but it is not well harmonised, particularly for selection of the highest dose tested. This paper further builds on concepts developed in a technical report by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) which recommends pragmatic approaches to dose selection considering regulatory requirements, animal welfare and state of the art scientific approaches. Industry sectors have differing degrees of freedom to operate regarding dose level selection, depending on the purpose of the studies and the regulatory requirements/legislation, and this is reflected in the overall recommended approaches. An understanding of systemic exposure should be utilised where possible (e.g., through toxicokinetic approaches) and used together with apical endpoints from existing toxicity studies to guide more appropriate dose level selection. The highest dose should be limited to a reasonable level, causing minimal but evident toxicity to the test animals without significantly compromising their well-being. As the science of predictive human exposure further develops and matures, this will provide exciting and novel opportunities for more human-relevant approaches to dose level selection.

Effect of Passing Plays on Injury Rates in the National Football League.

ABSTRACT: Burke, J, Geller, JS, Perez, JR, Naik, K, Vidal, AF, Baraga, MG, and Kaplan, LD. The effect of passing plays on injury rates in the national football league. J Strength Cond Res 35(12S): S1-S4, 2021-The National Football League (NFL) has one of the highest all-cause injury rates in sports, yet our understanding of extrinsic injury risk factors is limited. The objective of this study was to assess the effect of play type on injury incidence in the NFL. We obtained data for every regular season game played during the 2013-2016 seasons from the official NFL game books. There were 2,721 in-game injuries during the 4 seasons examined, with an overall rate of 1.33 injuries per team per game. For statistical analysis, p < 0.05 was considered significant. Passing plays conferred significantly higher odds of injury than running plays (odds ratio [OR] 1.4, 95% confidence interval [CI]: 1.3-1.5, p < 0.0001). This primarily stems from increased risks in quarterbacks (OR 6.9, 95% CI: 3.6-13.3, p < 0.0001), receivers (OR 5.0, 95% CI: 3.7-6.6, p < 0.0001), and defensive backs (OR 2.3, 95% CI: 1.9-2.7, p < 0.0001). Our study suggests that passing plays confer a greater risk of overall injuries in the NFL when compared with running plays, specifically regarding concussions and core or trunk injuries.

Hamstring injuries in the national football league: An epidemiological study.

Background: While numerous studies have evaluated National Football League injuries, there is limited literature evaluating hamstring injuries sustained in games. Our primary aim is to analyze the effect of player position on the relative incidence of hamstring injuries in the National Football League. Our secondary aims are to analyze the effects of field surface, week of the season, and short rest weeks. Methods: Official National Football League game books containing injury data from the 2013-2016 regular seasons were used. Data were analyzed to determine the incidence of hamstring injuries by field surface, rest, and week of the season. Field surface was considered either turf or grass. Short rest was considered four days. Relative incidence of hamstring injuries by position was performed with standardized incidence ratios. P values < 0.05 were considered statistically significant. Results: Seventy-eight qualifying hamstring injuries were identified and included in our analysis. Linebackers had the highest relative incidence per play with a standardized incidence ratio of 2.02 (CI: 1.14-2.91), followed by Defensive Backs (1.62; 95% CI: 1.14-1.62). Offensive linemen and defensive linemen had standardized incidence ratios significantly less than 1. Fifty-seven percent of hamstring injuries occurred on turf fields (p = 0.082). There was no significant difference between the proportion of hamstring injuries that occurred on short rest and the proportion of games played on short rest (p = 0.959). Hamstring injuries were not more likely to occur than the pooled group of all other types of injuries on short rest (p = 0.861). With a 17-week season, the mean week of hamstring injury was 8.05 (95% CI: 7.06-9.04), while the median week was 7.5. Conclusions: Linebackers and Defensive Backs have the highest relative incidence of hamstring injuries compared to other position groups, while offensive and defensive linemen have the lowest. Field surface and a short rest period did not show significance.

Persistent and interdependent: Racial disparities and their mechanisms in postmastectomy breast reconstruction.

BACKGROUND: Racial disparities in accessing postmastectomy breast reconstruction persist despite expansion of insurance coverage. An updated examination with a broad assessment of mediating factors in a "majority minority" community is needed. METHODS: Data were collected on all patients undergoing mastectomy for breast cancer from 2011 to 2019 in a private academic center and adjacent safety-net hospital. Multivariable logistic regression was used to assess the effect of race on postmastectomy breast reconstruction, controlling for predetermined potentially mediating and confounding variables. RESULTS: Of 1,554 patients, 63.8% (n = 203) of non-Hispanic White, 33.4% (n = 102) of Black, and 47.9% (n = 438) of Hispanic patients underwent postmastectomy breast reconstruction. Multivariable logistic regression showed that Black patients (odds ratio [OR] 3.6, 95% confidence internal [CI]: 2.2-5.9; P < .0001) undergo significantly less postmastectomy breast reconstruction than White patients. Age, insurance status, stage, and hospital type mediated this relationship. CONCLUSION: Black patients have substantially reduced rates of postmastectomy breast reconstruction compared with White patients, which is mediated by socioeconomic factors.

Stability of the domain interface contributes towards the catalytic function at the H-site of class alpha glutathione transferase A1-1.

Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the S(N)Ar reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain-domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs.

A Simple Risk Score to Predict Clavien-Dindo Grade IV and V Complications After Non-elective Cholecystectomy.

BACKGROUND: Non-elective cholecystectomies can lead to severe postoperative complications and mortality. Existing risk prediction tools do not meet the need to reliably predict these complications. METHODS: Using the 2011-2016 American College of Surgeons National Surgical Quality Improvement Program datasets, we identified patients undergoing non-elective cholecystectomy with primary ICD 9/10 codes indicating the following diagnoses: symptomatic cholelithiasis, acute cholecystitis, choledocholithiasis, gallstone pancreatitis, and cholangitis. We randomly allocated patients to derivation and validation cohorts (80/20 split). Severe complications (Clavien-Dindo grades IV and V) included unplanned intubation, prolonged mechanical ventilation, pulmonary embolism, acute renal failure requiring dialysis, stroke, myocardial infarction, cardiac arrest, septic shock, and mortality. Logistic regression using backward selection identified predictors of severe complications and a risk score was generated based on this model. RESULTS: Of 68,953 patients in the derivation cohort, 1.7% (N = 1157) suffered severe complications. The final multivariable risk score model included the following predictors: age (0-12 points), preoperative sepsis (5 points), planned open procedure (5 points), estimated glomerular filtration rate (0-13 points), and preoperative albumin level (0-8 points). The associated risk-score model yielded scores from 0 to 43 with 0.1-59.4% predicted probability of severe complications and had a C-statistic of 0.845 (95% CI 0.834, 0.857) in the derivation cohort and 0.870 (95% CI 0.851, 0.889) in the validation cohort. CONCLUSION: A simple risk-score model predicts severe complications in patients undergoing unplanned cholecystectomy for common indications encountered in an acute care surgery service and identifies high-risk patients.

The role of bronchoscopy in the diagnosis and management of pediatric pulmonary tuberculosis.

Bronchoscopy is useful as a diagnostic and therapeutic procedure in children with Tuberculosis (TB) disease complicated by airway obstruction. It is needed in children when surgical intervention may be required for airway compression, when drug resistance is suspected, and to rule out an alternative diagnosis for airway obstruction. Bronchoscopy with bronchoalveolar lavage (BAL) should be performed when other, less invasive samples cannot be collected, or when they fail to provide useful diagnostic information. BAL specimens collected at bronchoscopy can be tested using molecular TB assays and mycobacterial culture. The aim of this review is to evaluate the role of bronchoscopy in the diagnosis and management of pulmonary TB in children, and, specifically, to review the role of interventional bronchoscopy. A search of electronic databases was undertaken using the online databases PubMed, Ovid MEDLINE, EMBASE, Google Advanced Scholar, and Web of Science to identify relevant literature. The search was limited to pediatrics, pulmonology, bronchoscopy, and pediatric pulmonary tuberculosis for all articles published in English on pediatric bronchoscopy between 2010 and 2020. Recent advances in pediatric bronchoscopy was included, as well as recent research on improving the diagnosis with the use of interventional bronchoscopy. The role of bronchoscopy in pediatric pulmonary tuberculosis has changed during the last decade, from a simple method of collecting samples for bacteriological conformation to an more sophisticate procedure. New methods are available for collecting samples, which includes the use of Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and also better methods of bacteriological conformation. Interventions are now possible; not only to improve the diagnostic abilities of bronchoscopy but also to diagnose, manage, and follow-up upon airway-related complications. Bronchoscopy services remain limited in resource-limited countries due to the high cost of equipment.

Class Pi glutathione transferase unfolds via a dimeric and not monomeric intermediate: functional implications for an unstable monomer.

Cytosolic class pi glutathione transferase P1-1 (GSTP1-1) is associated with drug resistance and proliferative pathways because of its catalytic detoxification properties and ability to bind and regulate protein kinases. The native wild-type protein is homodimeric, and whereas the dimeric structure is required for catalytic functionality, a monomeric and not dimeric form of class pi GST is reported to mediate its interaction with and inhibit the activity of the pro-apoptotic enzyme c-Jun N-terminal kinase (JNK) [Adler, V., et al. (1999) EMBO J. 18, 1321-1334]. Thus, the existence of a stable monomeric form of wild-type class pi GST appears to have physiological relevance. However, there are conflicting accounts of the subunit's intrinsic stability since it has been reported to be either unstable [Dirr, H., and Reinemer, P. (1991) Biochem. Biophys. Res. Commun. 180, 294-300] or stable [Aceto, A., et al. (1992) Biochem. J. 285, 241-245]. In this study, the conformational stability of GSTP1-1 was re-examined by equilibrium folding and unfolding kinetics experiments. The data do not demonstrate the existence of a stable monomer but that unfolding of hGSTP1-1 proceeds via an inactive, nativelike dimeric intermediate in which the highly dynamic helix 2 is unfolded. Furthermore, molecular modeling results indicate that a dimeric GSTP1-1 can bind JNK. According to the available evidence with regard to the stability of the monomeric and dimeric forms of GSTP1-1 and the modality of the GST-JNK interaction, formation of a complex between GSTP1-1 and JNK most likely involves the dimeric form of the GST and not its monomer as is commonly reported.

The role of a topologically conserved isoleucine in glutathione transferase structure, stability and function.

The common fold shared by members of the glutathione-transferase (GST) family has a topologically conserved isoleucine residue at the N-terminus of helix 3 which is involved in the packing of helix 3 against two beta-strands in domain 1. The role of the isoleucine residue in the structure, function and stability of GST was investigated by replacing the Ile71 residue in human GSTA1-1 by alanine or valine. The X-ray structures of the I71A and I71V mutants resolved at 1.75 and 2.51 A, respectively, revealed that the mutations do not alter the overall structure of the protein compared with the wild type. Urea-induced equilibrium unfolding studies using circular dichroism and tryptophan fluorescence suggest that the mutation of Ile71 to alanine or valine reduces the stability of the protein. A functional assay with 1-chloro-2,4-dinitrobenzene shows that the mutation does not significantly alter the function of the protein relative to the wild type. Overall, the results suggest that conservation of the topologically conserved Ile71 maintains the structural stability of the protein but does not play a significant role in catalysis and substrate binding.

CIC-rearranged round cell (Ewing-like) sarcoma of the uterus: Review of the literature.

CIC-rearranged round cell sarcoma (CRS) is a rare entity that presents in various anatomical locations and involves deep soft-tissue structures and skin. Although commonly confused with and clinically similar to Ewing sarcoma (ES), investigators have recently shown that this unique condition maintains morphologic and pathologic features that are distinct from ES. In this report, we present and discuss a case of CRS of the uterus, the first of its kind to be reported in the English-language literature. We urge the scientific community to continue its investigations in elucidating the features of this entity, as young women who suffer from this condition have dismal prognoses and currently do not have access to therapeutic options for cure.

Bronchoscopy in children with COVID-19: A case series.

INTRODUCTION: The coronavirus disease-2019 (COVID-19) era is a challenging time for respiratory teams to protect their patients and staff. COVID-19 is predominantly transmitted by respiratory droplets; in the clinical setting, aerosol generating procedures pose the greatest risk for COVID-19 transmission. Bronchoscopy is associated with increased risk of patient-to-health care worker transmission, owing to aerosolized viral particles which may be inhaled and also result in environmental contamination of surfaces. METHODS: We describe our experience with the use of modified full-face snorkeling masks for pediatric bronchoscopy procedures in four COVID-19 infected children when filtering facepieces/respirators were in limited supply. RESULTS: Bronchoscopy was urgently required in four children, and could not be delayed until COVID-19 test results were available. During the pandemic peak, when respirators were in short supply, modified full-face snorkel masks (SEAC Libera, SEAC, Italy) were worn by the bronchoscopy team. Each mask was fitted with an O-ring, adapter, and heat and moisture exchanger filter. To date, there have been no COVID-19 infections among the bronchoscopy team staff, whereas the overall Hospital staff COVID-19 prevalence rate has exceeded 13.5% (667/4949). CONCLUSION: Emergency bronchoscopy procedures on COVID-19 infected patients or patients with unknown infection status can be safely performed using modified full-face snorkel masks.

The Effect of Thursday Night Games on In-Game Injury Rates in the National Football League.

BACKGROUND: Although claims of increased injury rates with Thursday night National Football League (NFL) games exist, a paucity of data exist substantiating these claims. PURPOSE: To evaluate the effect of rest between games on in-game injury rates as it pertains to overall injury incidence, location, and player position. STUDY DESIGN: Descriptive epidemiologic study. METHODS: Data were obtained from official NFL game books for regular season games from all 32 teams for the 2013-2016 seasons. All in-game injuries recorded in official game books were included. Rest periods between games were classified as short (4 days), regular (6-8 days), or long (≥10 days). Overall observed injury rates per team-game were analyzed in relation to different rest periods using negative binomial regression. For results with significant overall findings, pairwise comparisons were tested using the Wald chi-square test. Exploratory secondary analyses were performed in a similar fashion to assess differences in injury rates for the different rest periods when stratified by anatomic location and player position. RESULTS: A total of 2846 injuries were identified throughout the 4 seasons. There was an overall significant difference in injuries per team-game between short, regular, and long rest (P = .01). With short rest, an observed mean of 1.26 injuries per game (95% CI, 1.06-1.49) was significantly different from the 1.53 observed injuries per game with regular rest (95% CI, 1.46-1.60; P = .03), but not compared with the 1.34 observed injuries per game with long rest (P = .56). For player position, only the tight end, linebacker, and fullback group demonstrated significant differences between the injury rates for different rest categories. Quarterback was the only position with more injuries during games played on Thursday compared with both regular and long rest. This specific analysis was underpowered and the difference was not significant (P = .08). No differences were found regarding injury rates in correlation with differences in rest periods with different injury locations. CONCLUSION: A short rest period between games is not associated with increased rates of observed injuries reported in NFL game books; rather, our data suggest there are significantly fewer injuries for Thursday night games compared with games played on regular rest. Future research correlating rest and quarterback injury rates is warranted.

Effect of low salinity on cadmium accumulation and calcium homeostasis in the shore crab (Carcinus maenas) at fixed free Cd2+ concentrations.

Increased Cd toxicity at low salinity has been attributed to increased free Cd2+ ion concentration ([Cd2+]sw), but transfer to dilute seawater also stimulates physiological ionic regulation in crabs. In this study, Cd accumulation and Ca homeostasis in the shore crab (Carcinus maenas) were explored at fixed [Cd2+]sw to reveal the physiological events during sublethal Cd exposure. Crabs were exposed to 3.4 or 34 microg/L [Cd2+], in both 100% seawater (SW) and 33% SW for up to 10 d and sampled for hemolymph composition as well as gill and hepatopancreas Ca, Cd, and Ca-ATPase activity. Cadmium exposure ameliorated the expected fall in hemolymph osmotic pressure and NaCl at low salinity and generally protected tissue Ca from decline. Cadmium exposure alone (within salinity) inhibited Ca-ATPase, but this was offset by stimulation of Ca-ATPase at low salinity. The Ca-ATPase activity in the anterior and posterior gills showed different responses to Cd/low salinity stress. Crabs were more sensitive to a 10-fold increase in [Cd2+]sw at low salinity. Overall, we conclude that exposure to a fixed sublethal [Cd2+]sw reveals a compensatory physiological response that is driven primarily by salinity rather than Cd2+ free ion concentration. Physiological responses are therefore important during low-level Cd exposure in dilute seawater.

Arginine 15 stabilizes an S(N)Ar reaction transition state and the binding of anionic ligands at the active site of human glutathione transferase A1-1.

Arg15, conserved in class Alpha GSTs (glutathione transferases), is located at the interface between the G- and H-sites of the active site where its cationic guanidinium group might play a role in catalysis and ligand binding. Arg15 in human GSTA1-1 was replaced with a leucine and crystallographic, spectroscopic, thermodynamic and molecular docking methods were used to investigate the contribution made by Arg15 towards (i) the binding of glutathione (GSH) to the G-site, (ii) the pK(a) of the thiol group of GSH, (iii) the stabilization of an analog of the anionic transition state of the S(N)Ar reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, and, (iv) the binding of the anionic non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to the H-site. While the R15L mutation substantially diminishes the CDNB-GSH conjugating activity of the enzyme, it has little effect on protein structure and stability. Arg15 does not contribute significantly towards the enzyme's affinity for GSH but does determine the reactivity of GSH by reducing the thiol's pK(a) from 7.6 to 6.6. The anionic sigma-complex formed between GSH and 1,3,5-trinitrobenzene is stabilized by Arg15, suggesting that it also stabilizes the transition state formed in the S(N)Ar reaction between GSH and CDNB. The trinitrocyclohexadienate moiety of the sigma-complex binds the H-site where the catalytic residue, Tyr9, was identified to hydrogen bond to an o-nitro group of the sigma-complex. The affinity for ANS at the H-site is decreased about 3-fold by the R15L mutation implicating the positive electrostatic potential of Arg15 in securing the organic anion at this site.

Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer.

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.

Residue 219 impacts on the dynamics of the C-terminal region in glutathione transferase A1-1: implications for stability and catalytic and ligandin functions.

The C-terminal region in class alpha glutathione transferases (GSTs) modulates the catalytic and nonsubstrate ligand binding functions of these enzymes. Except for mouse GST A1-1 (mGST A1-1), the structures of class alpha GSTs have a bulky aliphatic side chain topologically equivalent to Ile219 in human GST A1-1 (hGST A1-1). In mGST A1-1, the corresponding residue is an alanine. To investigate the role of Ile219 in determining the conformational dynamics of the C-terminal region in hGST A1-1, the residue was replaced by alanine. The substitution had no effect on the global structure of hGST A1-1 but did reduce the conformational stability of the C-terminal region of the protein. This region could be stabilized by ligands bound at the active site. The catalytic behavior of hGST A1-1 was significantly compromised by the I219A mutation as demonstrated by reduced enzyme activity, increased K(m) for the substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), and reduced catalytic efficiencies. Inhibition studies also indicated that the binding affinities for product and substrate analogues were dramatically decreased. The affinity of the mutant for GSH was, however, only slightly increased, indicating that the G-site was unaltered by the mutation. The binding affinity and stoichiometry for the anionic dye 8-anilino-1-naphthalene sulfonate (ANS) was also not significantly affected by the I219A mutation. However, the lower DeltaC(p) for ANS binding to the mutant (-0.34 kJ/mol per K compared with -0.84 kJ/mol per K for the wild-type protein) suggests that ANS binding to the mutant results in the burial of less hydrophobic surface area. Fluorescence data also indicates that ANS bound to the mutant is more prone to quenching by water. Overall, the data from this study, together with the structural details of the C-terminal region in mGST A1-1, show that Ile219 is an important structural determinant of the stability and dynamics of the C-terminal region of hGST A1-1.

The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1.

The thioredoxin-like fold has a betaalphabetaalphabetabetaalpha topology, and most proteins/domains with this fold have a topologically conserved cis -proline residue at the N-terminus of beta-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347-350]. In order to further address the role of the cis -proline in the structure, function and stability of GSTs, cis -Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.