The mouse Gene Expression Database (GXD): 2017 update.

The Gene Expression Database (GXD; www.informatics.jax.org/expression.shtml) is an extensive and well-curated community resource of mouse developmental expression information. Through curation of the scientific literature and by collaborations with large-scale expression projects, GXD collects and integrates data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments. Expression data from both wild-type and mutant mice are included. The expression data are combined with genetic and phenotypic data in Mouse Genome Informatics (MGI) and made readily accessible to many types of database searches. At present, GXD includes over 1.5 million expression results and more than 300 000 images, all annotated with detailed and standardized metadata. Since our last report in 2014, we have added a large amount of data, we have enhanced data and database infrastructure, and we have implemented many new search and display features. Interface enhancements include: a new Mouse Developmental Anatomy Browser; interactive tissue-by-developmental stage and tissue-by-gene matrix views; capabilities to filter and sort expression data summaries; a batch search utility; gene-based expression overviews; and links to expression data from other species.

On the Ground or in the Air? A Methodological Experiment on Crop Residue Cover Measurement in Ethiopia.

Maintaining permanent coverage of the soil using crop residues is an important and commonly recommended practice in conservation agriculture. Measuring this practice is an essential step in improving knowledge about the adoption and impact of conservation agriculture. Different data collection methods can be implemented to capture the field level crop residue coverage for a given plot, each with its own implication on survey budget, implementation speed and respondent and interviewer burden. In this paper, six alternative methods of crop residue coverage measurement are tested among the same sample of rural households in Ethiopia. The relative accuracy of these methods are compared against a benchmark, the line-transect method. The alternative methods compared against the benchmark include: (i) interviewee (respondent) estimation; (ii) enumerator estimation visiting the field; (iii) interviewee with visual-aid without visiting the field; (iv) enumerator with visual-aid visiting the field; (v) field picture collected with a drone and analyzed with image-processing methods and (vi) satellite picture of the field analyzed with remote sensing methods. Results of the methodological experiment show that survey-based methods tend to underestimate field residue cover. When quantitative data on cover are needed, the best estimates are provided by visual-aid protocols. For categorical analysis (i.e., >30% cover or not), visual-aid protocols and remote sensing methods perform equally well. Among survey-based methods, the strongest correlates of measurement errors are total farm size, field size, distance, and slope. Results deliver a ranking of measurement options that can inform survey practitioners and researchers.

The mouse Gene Expression Database (GXD): 2014 update.

The Gene Expression Database (GXD; http://www.informatics.jax.org/expression.shtml) is an extensive and well-curated community resource of mouse developmental expression information. GXD collects different types of expression data from studies of wild-type and mutant mice, covering all developmental stages and including data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments. The data are acquired from the scientific literature and from researchers, including groups doing large-scale expression studies. Integration with the other data in Mouse Genome Informatics (MGI) and interconnections with other databases places GXD's gene expression information in the larger biological and biomedical context. Since the last report, the utility of GXD has been greatly enhanced by the addition of new data and by the implementation of more powerful and versatile search and display features. Web interface enhancements include the capability to search for expression data for genes associated with specific phenotypes and/or human diseases; new, more interactive data summaries; easy downloading of data; direct searches of expression images via associated metadata; and new displays that combine image data and their associated annotations. At present, GXD includes >1.4 million expression results and 250,000 images that are accessible to our search tools.

Distinct CD4+ helper T cells involved in primary and secondary responses to infection.

Helper T cells are critical for protective immunity, CD8(+) T-cell memory, and CD4(+) recall responses, but whether the same or distinct CD4(+) T cells are involved in these responses has not been established. Here we describe two CD4(+) T cells, LLO118 and LLO56, specific for an immunodominant Listeria monocytogenes epitope, with dramatically different responses to primary and secondary infection. Comparing in vivo responses, LLO118 T cells proliferate more strongly to primary infection, whereas surprisingly, LLO56 has a superior CD4(+) recall response to secondary infection. LLO118 T cells provide more robust help for CD8(+) T-cell responses to secondary infection than LLO56. We found no detectable differences in antigen sensitivity, but naive LLO118 T cells have much lower levels of CD5 and their T-cell receptor levels are dramatically down-regulated after their strong primary response. Thus, distinct CD4(+) helper T cells are specialized to help either in primary or secondary responses to infection.

Greater reduction in mid-treatment FDG-PET volume may be associated with worse survival in non-small cell lung cancer.

BACKGROUND AND PURPOSE: This study tested the hypotheses that 1) changes in mid-treatment fluorodeoxyglucose (FDG)-positron emission tomography (PET) parameters are predictive of overall survival (OS) and 2) mid-treatment FDG-PET-adapted treatment has the potential to improve survival in patients with non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: Patients with stage I-III NSCLC requiring daily fractionated radiation were eligible. FDG-PET-CT scans were obtained prior to and mid-treatment with radiotherapy at 40-50 Gy. The normalized maximum standardized uptake value (NSUVmax), normalized mean SUV (NSUVmean), PET-metabolic tumor volume (MTV), total lesion glycolysis (TLG), and computed tomography-based gross tumor volume (CT-GTV) were consistently measured for all patients. The primary study endpoint was OS. RESULTS: The study is comprised of 102 patients who received 3-dimensional conformal radiotherapy, among whom 30 patients who received mid-treatment PET-adapted dose escalation radiotherapy. All PET-CT parameters decreased significantly (P < 0.001) mid-treatment, with greater reductions in FDG-volumetric parameters compared to FDG-activity factors. Mid-treatment changes in MTV (P = 0.053) and TLG (P = 0.021) were associated with OS, while changes in NSUVmax, NSUVmean, and CT-GTV were not (all Ps>0.1). Patients receiving conventional radiation (60-70 Gy) with MTV reductions greater than the mean had a median survival of 14 months, compared to those with MTV reductions less than the mean who had a median survival of 22 months. By contrast, patients receiving mid-treatment PET-adapted radiation with MTV reductions greater than the mean had a median survival of 33 months, compared to those with MTV reductions less than the mean who had a median survival of 19 months. Overall, PET-adapted treatment resulted in a 19% better 5-year survival than conventional radiation. CONCLUSION: Changes in mid-treatment PET-volumetric parameters were significantly associated with survival in NSCLC. A greater reduction in the mid-treatment MTV was associated with worse survival in patients treated with standard radiation, but with better survival in patients who received mid-treatment PET-adapted treatment.

Complete excision or marsupialization of ureteroceles: does choice of surgical approach affect outcome?

PURPOSE: Two accepted open surgical techniques exist for lower urinary tract reconstruction for ureteroceles, that is complete excision/enucleation and marsupialization/partial excision. To our knowledge it is currently unknown whether 1 method offers better clinical outcomes. We sought to answer this question. MATERIALS AND METHODS: We retrospectively reviewed the records of patients who underwent open surgical repair for ureterocele at 3 academic institutions. The 2 groups (complete excision vs marsupialization) were compared for each clinical outcome, including ongoing vesicoureteral reflux, new bladder diverticulum, hydronephrosis, continence, urinary tract infection and voiding dysfunction. Each clinical outcome was analyzed for independence from the surgical method. RESULTS: A total of 33 cases of complete excision and 24 of marsupialization were collected. The excision and marsupialization groups were similar with respect to patient age at surgery, gender and average followup (55 and 38 months, respectively). Most ureteroceles were unilateral and part of a duplicated system. Approximately half were intravesical and vesicoureteral reflux was the primary indication for surgery. In the excision group 82% of ureteroceles were previously punctured but only 50% were punctured in the marsupialization group (p = 0.09). Postoperatively the groups did not differ significantly in terms of ongoing vesicoureteral reflux (13% and 14%), bladder diverticulum (3% and 9%), continence (100% and 95%), urinary tract infection (43% and 46%) or voiding dysfunction (24% and 25%, respectively). They varied significantly in terms of improved or stable hydronephrosis (70% vs 91%, p = 0.05). CONCLUSIONS: When performing lower urinary tract reconstruction for ureterocele, this study demonstrates that the choice of complete excision vs marsupialization/partial excision does not appear to differentially affect clinical outcomes.

A model combining age, equivalent uniform dose and IL-8 may predict radiation esophagitis in patients with non-small cell lung cancer.

BACKGROUND AND PURPOSE: To study whether cytokine markers may improve predictive accuracy of radiation esophagitis (RE) in non-small cell lung cancer (NSCLC) patients. MATERIALS AND METHODS: A total of 129 patients with stage I-III NSCLC treated with radiotherapy (RT) from prospective studies were included. Thirty inflammatory cytokines were measured in platelet-poor plasma samples. Logistic regression was performed to evaluate the risk factors of RE. Stepwise Akaike information criterion (AIC) and likelihood ratio test were used to assess model predictions. RESULTS: Forty-nine of 129 patients (38.0%) developed grade ≥2 RE. Univariate analysis showed that age, stage, concurrent chemotherapy, and eight dosimetric parameters were significantly associated with grade ≥2 RE (p < 0.05). IL-4, IL-5, IL-8, IL-13, IL-15, IL-1α, TGFα and eotaxin were also associated with grade ≥2 RE (p < 0.1). Age, esophagus generalized equivalent uniform dose (EUD), and baseline IL-8 were independently associated grade ≥2 RE. The combination of these three factors had significantly higher predictive power than any single factor alone. Addition of IL-8 to toxicity model significantly improves RE predictive accuracy (p = 0.019). CONCLUSIONS: Combining baseline level of IL-8, age and esophagus EUD may predict RE more accurately. Refinement of this model with larger sample sizes and validation from multicenter database are warranted.

Systemic Levels of Estrogens and PGE2 Synthesis in Relation to Postmenopausal Breast Cancer Risk.

Background: Prostaglandin E2 (PGE2) induces aromatase expression in adipose tissue, leading to increased estrogen production that may promote the development and progression of breast cancer. However, few studies have simultaneously investigated systemic levels of PGE2 and estrogen in relation to postmenopausal breast cancer risk.Methods: Here, we determined urinary estrogen metabolites (EM) using mass spectrometry in a case-cohort study (295 incident breast cancer cases and 294 subcohort members), and using linear regression estimated the effect of urinary levels of a major PGE2 metabolite (PGE-M) on EMs. HRs for the risk of developing breast cancer in relation to PGE-M and EMs were compared between Cox regression models with and without mutual adjustment.Results: PGE-M was a significant predictor of estrone (E1), but not estradiol (E2) levels in multivariable analysis. Elevated E2 levels were associated with an increased risk of developing breast cancer [HRQ5vs.Q1, 1.54; 95% confidence interval (CI), 1.01-2.35], and this association remained unchanged after adjustment for PGE-M (HRQ5vs.Q1, 1.52; 95% CI, 0.99-2.33). Similarly, elevated levels of PGE-M were associated with increased risk of developing breast cancer (HRQ4vs.Q1, 2.01; 95% CI, 1.01-4.29), and this association was only nominally changed after consideration of E1 or E2 levels.Conclusions: Urinary levels of PGE-M and estrogens were independently associated with future risk of developing breast cancer among these postmenopausal women.Impact: Increased breast cancer risk associated with PGE-M might not be fully explained by the estrogens-breast cancer association alone but also by additional effects related to inflammation. Cancer Epidemiol Biomarkers Prev; 26(3); 383-8. ©2016 AACR.

Long Acting Liposomal Bupivacaine for Percutaneous Sympathetic Stellate Ganglion Blockade: A Technical Note.

INTRODUCTION: We describe the use of long acting liposomal bupivacaine for percutaneous stellate ganglion blockade to treat severe headaches following internal carotid artery dissection. METHODS: A 43-year old woman developed right-sided refractory headache after right internal carotid artery dissection. Patient underwent percutaneous stellate ganglion block using bupivacaine hydrochloride (0.25%-20 ml) in the past with short acting relief. Liposomal bupivacaine (EXPAREL) 13.3 mg/mL (1.3%) solution diluted with preservative-free normal saline: a total solution of 20 ml (52 mg of bupivacaine) was injected at the level of the lower portion of body of the sixth cervical vertebra, medial to the right internal carotid artery. The response to sympathetic block was assessed by a neurologist not involved in the procedure. RESULTS: After the stellate ganglion block with bupivacaine hydrochloride, patient was headache free immediately after the block but with recurrence of pain on Day 3 with return to peak intensity by Day 4. After the stellate ganglion blockade with liposomal bupivacaine hydrochloride, patient reported recurrence of pain on Day 15 post injection with return to peak intensity by Day 17. The patient reported an episode of aura which consisted of visual scintillations on Day 2 which lasted for five days and resolved spontaneously. CONCLUSION: Liposomal bupivacaine injection for stellate ganglion blockade can result in a more prolonged effect compared with bupivacaine hydrochloride.

Plasma Levels of IL-8 and TGF-ß1 Predict Radiation-Induced Lung Toxicity in Non-Small Cell Lung Cancer: A Validation Study.

PURPOSE AND OBJECTIVES: We previously reported that the combination of mean lung dose (MLD) and inflammatory cytokines interleukin-8 (IL-8) and transforming growth factor-ß1 (TGF-ß1) may provide a more accurate model for radiation-induced lung toxicity (RILT) prediction in 58 patients with non-small cell lung cancer (NSCLC). This study is to validate the previous findings with new patients and to explore new models with more cytokines. METHODS AND MATERIALS: One hundred forty-two patients with stage I-III NSCLC treated with definitive radiation therapy (RT) from prospective studies were included. Sixty-five new patients were used to validate previous findings, and all 142 patients were used to explore new models. Thirty inflammatory cytokines were measured in plasma samples before RT and 2 weeks and 4 weeks during RT (pre, 2w, 4w). Grade ≥2 RILT was defined as grade 2, and higher radiation pneumonitis or symptomatic pulmonary fibrosis was the primary endpoint. Logistic regression was performed to evaluate the risk factors of RILT. The area under the curve (AUC) for the receiver operating characteristic curves was used for model assessment. RESULTS: Sixteen of 65 patients (24.6%) experienced RILT2. Lower pre IL-8 and higher TGF-ß1 2w/pre ratio were associated with higher risk of RILT2. The AUC increased to 0.73 by combining MLD, pre IL-8, and TGF-ß1 2w/pre ratio compared with 0.61 by MLD alone to predict RILT. In all 142 patients, 29 patients (20.4%) experienced grade ≥2 RILT. Among the 30 cytokines measured, only IL-8 and TGF-ß1 were significantly associated with the risk of RILT2. MLD, pre IL-8 level, and TGF-ß1 2w/pre ratio were included in the final predictive model. The AUC increased to 0.76 by combining MLD, pre IL-8, and TGF-ß1 2w/pre ratio compared with 0.62 by MLD alone. CONCLUSIONS: We validated that a combination of mean lung dose, pre IL-8 level, and TGF-ß1 2w/pre ratio provided a more accurate model to predict the risk of RILT2 compared with MLD alone.

The Pretreatment Neutrophil/Lymphocyte Ratio Is Associated with All-Cause Mortality in Black and White Patients with Non-metastatic Breast Cancer.

The pretreatment neutrophil/lymphocyte ratio (NLR), derived from differential white blood cell counts, has been previously associated with poor prognosis in breast cancer. Little data exist, however, concerning this association in Black patients, who are known to have lower neutrophil counts than other racial groups. We conducted a retrospective cohort study of 236 Black and 225 non-Hispanic White breast cancer patients treated at a single institution. Neutrophil and lymphocyte counts were obtained from electronic medical records. Univariate and multivariate Cox regression models were used to determine hazard ratios (HRs) and 95% confidence intervals (95% CIs) of all-cause mortality and breast cancer-specific mortality in relation to pretreatment NLR. Overall, there were no associations between an elevated pretreatment NLR (NLR ≥3.7) and all-cause or breast cancer-specific mortality. Among patients without metastasis at the time of diagnosis, an elevated pretreatment NLR was independently associated with all-cause mortality, with a multivariable HR of 2.31 (95% CI: 1.10-4.86). Black patients had significantly lower NLR values than White patients, but there was no evidence suggesting racial heterogeneity of the prognostic utility of NLR. Pretreatment NLR was an independent predictor of all-cause mortality but not breast cancer-specific mortality in non-metastatic breast cancer patients.

Treatment-Related Death during Concurrent Chemoradiotherapy for Locally Advanced Non-Small Cell Lung Cancer: A Meta-Analysis of Randomized Studies.

Treatment related death (TRD) is the worst adverse event in chemotherapy and radiotherapy for patients with cancer, the reports for TRDs were sporadically. We aimed to study TRDs in non-small cell lung cancer (NSCLC) patients treated with concurrent chemoradiotherapy (CCRT), and determine whether high radiation dose and newer chemotherapy regimens were associated with the risk of TRD. Data from randomized clinical trials for locally advanced/unresectable NSCLC patients were analyzed. Eligible studies had to have at least one arm with CCRT. The primary endpoint was TRD. Pooled odds ratios (ORs) for TRDs were calculated. In this study, a total of fifty-three trials (8940 patients) were eligible. The pooled TRD rate (accounting for heterogeneity) was 1.44% for all patients. In 20 trials in which comparison of TRDs between CCRT and non-CCRT was possible, the OR (95% CI) of TRDs was 1.08 (0.70-1.66) (P = 0.71). Patients treated with third-generation chemotherapy and concurrent radiotherapy had an increase of TRDs compared to those with other regimens in CCRT (2.70% vs. 1.37%, OR = 1.50, 95% CI: 1.09-2.07, P = 0.008). No significant difference was found in TRDs between high (≥ 66 Gy) and low (< 66 Gy) radiation dose during CCRT (P = 0.605). Neither consolidation (P = 0.476) nor induction chemotherapy (P = 0.175) had significant effects with increased TRDs in this study. We concluded that CCRT is not significantly associated with the risk of TRD compared to non-CCRT. The third-generation chemotherapy regimens may be a risk factor with higher TRDs in CCRT, while high dose radiation is not significantly associated with more TRDs. This observation deserves further study.

Identification of a functionally important negatively charged residue within the second catalytic site of the SUR1 nucleotide-binding domains.

The ATP-sensitive K+ channel (KATP channel) couples glucose metabolism to insulin secretion in pancreatic beta-cells. It is comprised of sulfonylurea receptor (SUR)-1 and Kir6.2 proteins. Binding of Mg nucleotides to the nucleotide-binding domains (NBDs) of SUR1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. To elucidate the structural basis of this regulation, we constructed a molecular model of the NBDs of SUR1, based on the crystal structures of mammalian proteins that belong to the same family of ATP-binding cassette transporter proteins. This model is a dimer in which there are two nucleotide-binding sites, each of which contains residues from NBD1 as well as from NBD2. It makes the novel prediction that residue D860 in NBD1 helps coordinate Mg nucleotides at site 2. We tested this prediction experimentally and found that, unlike wild-type channels, channels containing the SUR1-D860A mutation were not activated by MgADP in either the presence or absence of MgATP. Our model should be useful for designing experiments aimed at elucidating the relationship between the structure and function of the KATP channel.

Nucleotide binding to the homodimeric MJ0796 protein: a computational study of a prokaryotic ABC transporter NBD dimer.

Transport by ABC proteins requires a cycle of ATP-driven conformational changes of the nucleotide binding domains (NBDs). We compare three molecular dynamics simulations of dimeric MJ0796: with ATP was present at both NBDs; with ATP at one NBD but ADP at the other; and without any bound ATP. In the simulation with ATP present at both NBDs, the dimeric protein interacts with the nucleotides in a symmetrical manner. However, if ADP is present at one binding site then both NBD-NBD and protein-ATP interactions are enhanced at the opposite site.

An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulfide cross-linking and homology modeling.

The multidrug resistance P-glycoprotein mediates the extrusion of chemotherapeutic drugs from cancer cells. Characterization of the drug binding and ATPase activities of the protein have made it the paradigm ATP binding cassette (ABC) transporter. P-glycoprotein has been imaged at low resolution by electron cryo-microscopy and extensively analyzed by disulphide cross-linking, but a high resolution structure solved ab initio remains elusive. Homology models of P-glycoprotein were generated using the structure of a related prokaryotic ABC transporter, the lipid A transporter MsbA, as a template together with structural data describing the dimer interface of the nucleotide binding domains (NBDs). The first model, which maintained the NBD:transmembrane domain (TMD) interface of MsbA, did not satisfy previously published cross-linking data. This suggests that either P-glycoprotein has a very different structure from MsbA or that the published E. coli MsbA structure does not reflect a physiological state. To distinguish these alternatives, we mapped the interface between the two TMDs of P-glycoprotein experimentally by chemical cross-linking of introduced triple-cysteine residues. Based on these data, a plausible atomic model of P-glycoprotein could be generated using the MsbA template, if the TMDs of MsbA are reoriented with respect to the NBDs. This model will be important for understanding the mechanism of P-glycoprotein and other ABC transporters.

Membrane protein structure quality in molecular dynamics simulation.

Our goal was to assess the relationship between membrane protein quality, output from protein quality checkers and output from molecular dynamics (MD) simulations. Membrane transport proteins are essential for a wide range of cellular processes. Structural features of integral membrane proteins are still under-explored due to experimental limitations in structure determination. Computational techniques can be used to exploit biochemical and medium resolution structural data, as well as sequence homology to known structures, and enable us to explore the structure-function relationships in several transmembrane proteins. The quality of the models produced is vitally important to obtain reliable predictions. An examination of the relationship between model stability in molecular dynamics (MD) simulations derived from RMSD (root mean squared deviation) and structure quality assessment from various protein quality checkers was undertaken. The results were compared to membrane protein structures, solved at various resolution, by either X-ray or electron diffraction techniques. The checking programs could predict the potential success of MD in making functional conclusions. MD stability was shown to be a good indicator for the quality of structures. The quality was also shown to be dependent on the resolution at which the structures were determined.

Dynamics and function in a bacterial ABC transporter: simulation studies of the BtuCDF system and its components.

ABC transporters are integral membrane proteins which couple the energy of ATP hydrolysis to the translocation of solutes across cell membranes. BtuCD is a approximately 1100-residue protein found in the inner membrane of Gram-negative bacteria which transports vitamin B12. Vitamin B12 is bound in the periplasm by BtuF, which delivers the solute to the periplasmic entrance of the transporter protein complex BtuCD. Molecular dynamics simulations of the BtuCD and BtuCDF complexes (in a lipid bilayer) and of the isolated BtuD and BtuF proteins (in water) have been used to explore the conformational dynamics of this complex transport system. Overall, seven simulations have been performed, with and without bound ATP, corresponding to a total simulation time of 0.1 micros. Binding of ATP drives closure of the nucleotide-binding domains (NBDs) in BtuD in a symmetrical fashion, but not in BtuCD. It seems that ATP constrains the flexibility of the NBDs in BtuCD such that their closure may only occur upon binding of BtuF to the complex. Upon introduction of BtuF, and concomitant with NBD association, one ATP-binding site displays a closure, while the opposite site remains relatively unchanged. This asymmetry may reflect an initial step in the "alternating hydrolysis" mechanism and is consistent with measurements of nucleotide-binding stoichiometries. Principal components analysis of the simulation of BtuCD reveals motions that are comparable to those suggested in current transport models.

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein.

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.