Switch I closure simultaneously promotes strong binding to actin and ADP in smooth muscle myosin.

The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites.

Proteomic analysis of resectable non-small cell lung cancer: post-resection serum samples may be useful in identifying potential markers.

Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) can be used to analyse peptides and proteins in clinical samples. A prospective study was undertaken on patients undergoing curative resection for non-small cell lung cancer (NSCLC): we used SELDI-TOF-MS to compare the proteomic profiles of serum from these patients both before surgical resection and after resection (disease-free) to identify potential biomarkers. Student t-tests were used, and a P-value of <0.

Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity.

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm.

Applications of phasors to in vitro time-resolved fluorescence measurements.

The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions such as solvent relaxation and Förster resonance energy transfer. The method uses a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principal advantage of the phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection.

Applications of phasor plots to in vitro protein studies.

In a recent article, we described the application of phasor analysis to fluorescence intensity decay data on in vitro samples. As detailed in that article, this method provides researchers with a simple graphical method for viewing lifetime data that can be used to quantify individual components of a mixture as well as to identify excited state reactions. In the current article, we extend the use of in vitro phasor analysis to intrinsic protein fluorescence.

Final safety and efficacy analysis of the specific endothelin A receptor antagonist zibotentan (ZD4054) in patients with metastatic castration-resistant prostate cancer and bone metastases who were pain-free or mildly symptomatic for pain: a double-blind, placebo-controlled, randomized Phase II trial.

Objectives: To report the final analysis of a Phase II trial, which investigated the safety and efficacy of the specific endothelin A receptor antagonist zibotentan (AstraZeneca, Macclesfield, UK) in patients with metastatic castration-resistant prostate cancer (CRPC).

Patients And Methods: Patients with CRPC and bone metastases who were pain free or mildly symptomatic for pain were randomized to receive once-daily oral tablets of zibotentan 10 mg, 15 mg or placebo. The primary endpoint was the time to progression and secondary endpoints included overall survival, change in the number of bone metastases, and safety.

Sunitinib in patients with metastatic renal cell carcinoma: Birmingham experience.

Sunitinib is a novel, multi-targeted receptor tyrosine kinase inhibitor, which has demonstrated evidence of improved survival when compared to interferon (IFN)-alpha in patients with metastatic renal cell carcinoma (RCC). Recently published National Institute for Health and Clinical Excellence guidance recommends sunitinib as a first-line treatment option for patients with advanced and/or metastatic RCC. We assessed the efficacy and toxicity of sunitinib in an unselected group of patients with metastatic RCC, and compared outcomes in clinical practice with published clinical trial results.

Interferon alfa-2a versus combination therapy with interferon alfa-2a, interleukin-2, and fluorouracil in patients with untreated metastatic renal cell carcinoma (MRC RE04/EORTC GU 30012): an open-label randomised trial.

Background: In metastatic renal cell carcinoma combinations of interferon alfa-2a, interleukin-2, and fluorouracil produce higher response rates and longer progression-free survival than do single agents. We aimed to compare overall survival in patients receiving combination treatment or interferon alfa-2a.

Methods: RE04/30012 was an open-label randomised trial undertaken in 50 centres across eight countries.

Excited-state lifetime studies of the three tryptophan residues in the N-lobe of human serum transferrin.

The energy transfer from the three Trp residues at positions 8, 128, and 264 within the human serum transferrin (hTF) N-lobe to the ligand to metal charge transfer band has been investigated by monitoring changes in Trp fluorescence emission and lifetimes. The fluorescence emission from hTF N-lobe is dominated by Trp264, as revealed by an 82% decrease in the quantum yield when this Trp residue is absent. Fluorescence lifetimes were determined by multifrequency phase fluorometry of mutants containing one or two Trp residues.

Multinational, double-blind, phase III study of prednisone and either satraplatin or placebo in patients with castrate-refractory prostate cancer progressing after prior chemotherapy: the SPARC trial.

Purpose: This multinational, double-blind, randomized, placebo-controlled, phase III trial assessed the efficacy and tolerability of the oral platinum analog satraplatin in patients with metastatic castrate-refractory prostate cancer (CRPC) experiencing progression after one prior chemotherapy regimen.

Patients And Methods: Nine hundred fifty patients were randomly assigned (2:1) to receive oral satraplatin (n = 635) 80 mg/m(2) on days 1 to 5 of a 35-day cycle and prednisone 5 mg twice daily or placebo (n = 315) and prednisone 5 mg twice daily. Primary end points were progression-free survival and overall survival (OS).

Biomarkers in bladder cancer.

Detailed molecular insights into bladder cancer biology might allow more detailed prognostication and optimization of treatment with the objective of improving patient outcome and quality of life. However, in bladder cancer research the search for biomarkers has been called into question and has even obtained notoriety. It is unlikely that any single marker will be able to improve prognostication for patients with bladder cancer above and beyond grade and stage, but a combination of multiple independent markers might more precisely predict the outcome.

Equine transcriptome quantification using human GeneChip arrays can be improved using genomic DNA hybridisation and probe selection.

Affymetrix GeneChip arrays are a powerful tool for transcriptome profiling and have been applied to a wide range of species. A genomic DNA (gDNA)-based probe selection method has been developed which broadens the range of species to which GeneChips may be successfully applied. This study demonstrated that gDNA-based probe selection on the Affymetrix U133+2 GeneChip array can be used to study the equine transcriptome which, to date, has received only limited attention.

Issues in applying multi-arm multi-stage methodology to a clinical trial in prostate cancer: the MRC STAMPEDE trial.

Background: The multi-arm multi-stage (MAMS) trial is a new paradigm for conducting randomised controlled trials that allows the simultaneous assessment of a number of research treatments against a single control arm. MAMS trials provide earlier answers and are potentially more cost-effective than a series of traditionally designed trials. Prostate cancer is the most common tumour in men and there is a need to improve outcomes for men with hormone-sensitive, advanced disease as quickly as possible.

A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954 [correction of CB1984].

We report a phase I/II clinical trial in prostate cancer (PCa) using direct intraprostatic injection of a replication defective adenovirus vector (CTL102) encoding bacterial nitroreductase (NTR) in conjunction with systemic prodrug CB1954. One group of patients with localized PCa scheduled for radical prostatectomy received virus alone, prior to surgery, in a dose escalation to establish safety, tolerability, and NTR expression. A second group with local failure following primary treatment received virus plus prodrug to establish safety and tolerability.

Inequivalent contribution of the five tryptophan residues in the C-lobe of human serum transferrin to the fluorescence increase when iron is released.

Human serum transferrin (hTF), with two Fe3+ binding lobes, transports iron into cells. Diferric hTF preferentially binds to a specific receptor (TFR) on the surface of cells, and the complex undergoes clathrin dependent receptor-mediated endocytosis. The clathrin-coated vesicle fuses with an endosome where the pH is lowered, facilitating iron release from hTF.

Structural and functional consequences of the substitution of glycine 65 with arginine in the N-lobe of human transferrin.

The G65R mutation in the N-lobe of human transferrin was created to mimic a naturally occurring variant (G394R) found in the homologous C-lobe. Because Gly65 is hydrogen-bonded to the iron-binding ligand Asp63, it comprises part of the second-shell hydrogen bond network surrounding the iron within the metal-binding cleft of the protein. Substitution with an arginine residue at this position disrupts the network, resulting in much more facile removal of iron from the G65R mutant.

Sorafenib induces therapeutic response in a patient with metastatic collecting duct carcinoma of kidney.

Background: Collecting duct carcinoma (CDC) is a rare and aggressive variant of renal cell carcinoma (RCC), which has poor response to cytokine therapy and chemotherapy. Introduction of tyrosine kinase inhibitors (TKIs), in particular sorafenib and sunitinib, is changing the treatment paradigm for management of RCC. However, patients with CDC have been excluded from the majority of randomised trials involving the use of TKIs.

Incorporation of 5-hydroxytryptophan into transferrin and its receptor allows assignment of the pH induced changes in intrinsic fluorescence when iron is released.

Human serum transferrin (hTF) is a bilobal glycoprotein that transports iron to cells. At neutral pH, diferric hTF binds with nM affinity to the transferrin receptor (TFR) on the cell surface. The complex is taken into the cell where, at the acidic pH of the endosome ( approximately pH 5.

Safety and efficacy of the specific endothelin-A receptor antagonist ZD4054 in patients with hormone-resistant prostate cancer and bone metastases who were pain free or mildly symptomatic: a double-blind, placebo-controlled, randomised, phase 2 trial.

Background: The endothelin-A receptor (ETAR) has been implicated in the progression of prostate cancer.

Objectives: To investigate the safety and efficacy of the specific ETAR antagonist ZD4054 in patients with metastatic hormone-resistant prostate cancer (HRPC).

Design, Setting, And Participants: Double-blind, placebo-controlled, randomised, parallel-group, multicentre, phase 2 trial in patients attending cancer centres with HRPC and bone metastases who were pain free or mildly symptomatic for pain.

Systemic therapy for advancing or metastatic prostate cancer (STAMPEDE): a multi-arm, multistage randomized controlled trial.

There is a need to improve the outcomes for men with high-risk localised, nodal or metastatic prostate cancer, or with aggressively relapsing disease after initial therapy for local disease. This group of men is currently managed with long-term hormone therapy. Thus we aim to evaluate the toxicity and efficacy of three different systemic therapies (docetaxel, zoledronic acid and celecoxib) used alone or combined at the initiation of hormone manipulation for high-risk prostate cancer.

Speeding up the evaluation of new agents in cancer.

Despite both the increase in basic biologic knowledge and the fact that many new agents have reached various stages of development during the last 10 years, the number of new treatments that have been approved for patients has not increased as expected. We propose the multi-arm, multi-stage trial design as a way to evaluate treatments faster and more efficiently than current standard trial designs. By using intermediate outcomes and testing a number of new agents (and combinations) simultaneously, the new design requires fewer patients.