Serum proteomic test in advanced non-squamous non-small cell lung cancer treated in first line with standard chemotherapy.

BACKGROUND: VeriStrat is a blood-based proteomic test with predictive and prognostic significance in second-line treatments for non-small cell lung cancer (NSCLC). This trial was designed to investigate the role of VeriStrat in first-line treatment of advanced NSCLC with standard chemotherapy. Here we present the results for 76 non-squamous patients treated with a combination of carboplatin or cisplatin with pemetrexed. METHODS: The test-assigned classifications of VeriStrat Good or VeriStrat Poor to samples collected at baseline. The primary end point was progression-free survival (PFS); secondary end points included overall survival (OS) and objective response. Exploratory analyses of end points separately in carboplatin/pemetrexed and cisplatin/pemetrexed subgroups were also conducted. RESULTS: Patients classified as VeriStrat Good had longer PFS and OS than VeriStrat Poor: 6.5 vs 1.6 months and 10.8 vs 3.4 months, respectively; the corresponding hazard ratios (HRs) were 0.36 (P<0.0001) and 0.26 (P<0.0001); they were also more likely to achieve objective response. Prognostic significance of VeriStrat was confirmed in multivariate analysis. Significant differences in OS and PFS between Veristrat classifications were also found when treatment subgroups were analysed separately. CONCLUSIONS: The trial demonstrated clinical utility of VeriStrat as a prognostic test for standard first-line chemotherapy of non-squamous advanced NSCLC.

VeriStrat(®) has prognostic value in advanced stage NSCLC patients treated with erlotinib and sorafenib.

BACKGROUND: The serum proteomic test VeriStrat has been shown to be able to classify advanced non-small cell lung cancer (NSCLC) patients for overall survival (OS) after treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). In this study, VeriStrat was evaluated as a pre-treatment stratification tool in patients with advanced stage NSCLC for treatment with the combination of erlotinib and sorafenib, considering both OS and progression-free survival (PFS) as end points. METHODS: Serum samples from 50 patients treated within the context of a phase II trial of first-line erlotinib and sorafenib were analysed with VeriStrat, a fully locked mass spectrometry-based test that identifies patients likely to have good or poor outcome on EGFR therapy based on eight distinct features in mass spectra. Analysis was performed fully blinded to all clinical data, and then the outcome data were analysed with respect to the obtained serum classifications. RESULTS: VeriStrat classified pre-treatment samples into two groups, VeriStrat Good and VeriStrat Poor, which were significantly different in OS (hazard ratio (HR) 0.30, log-rank P=0.009) and in PFS (HR 0.40, log-rank P=0.035). CONCLUSION: VeriStrat has shown its potential for stratification of unselected, advanced stage NSCLC patients treated in first line with a combination of erlotinib and sorafenib.

An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR.

The interaction of a protein antigen, horse cytochrome c (cyt c), with a monoclonal antibody has been studied by hydrogen-deuterium (H-D) exchange labeling and two-dimensional nuclear magnetic resonance (2D NMR) methods. The H-exchange rate of residues in three discontiguous regions of the cyt c polypeptide backbone was slowed by factors up to 340-fold in the antibody-antigen complex compared with free cyt c. The protected residues, 36 to 38, 59, 60, 64 to 67, 100, and 101, and their hydrogen-bond acceptors, are brought together in the three-dimensional structure to form a contiguous, largely exposed protein surface with an area of about 750 square angstroms. The interaction site determined in this way is consistent with prior epitope mapping studies and includes several residues that were not previously identified. The hydrogen exchange labeling approach can be used to map binding sites on small proteins in antibody-antigen complexes and may be applicable to protein-protein and protein-ligand interactions in general.

Kinetic role of early intermediates in protein folding.

The traditional view that partly folded intermediates are important for directing a protein toward the native state has been challenged by the notion that proteins can intrinsically fold rapidly in a single step if kinetic complications due to slow conformational events are avoided. Intermediates that accumulate within the first few milliseconds of folding are, however, a common observation even for small single-domain proteins. Recent spectroscopic studies, coupled with quantitative kinetic analysis, suggest that folding is facilitated by the rapid formation of compact intermediates with some native-like structural features.

Methods for exploring early events in protein folding.

Progress in understanding dynamic aspects of protein folding relies on the continuing development of methods for obtaining more detailed structural information on the transient conformational ensembles that often appear within microseconds of initiating refolding. Advances in rapid mixing and other time-resolved spectroscopic methods have made it possible to explore some of the earliest stages of folding, including the initial formation of compact states, which is determined by the presence of a sequence-specific kinetic barrier, as well as the 'downhill' folding kinetics after the rate-limiting barrier has been crossed.

Changes in dendritic structure and function following hippocampal lesions: correlations with developmental events?

Recovery after nervous system lesions may lead to partial re-institution of developmental schemes and processes. Here we review several of these proposed schemes, with the conclusion that though some processes may involve re-expression of embryonic phenotypes, there are many processes invoked during recovery from lesions that do not mirror developmental phenomena. The inability to fully revert to embryonic schemes because of adult phenotype may partially account for the decreased recovery observed in adults compared to that noted after lesions during development.

Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics.

The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like alpha-helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-(2)H exchange using two-dimensional NMR spectroscopy and electrospray ionisation mass spectrometry. The H-(2)H exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1(+/-0.5) h(-1), which reflects the rate of transition from the lipid-inserted state, H(l), to an unprotected conformation, D(i), associated with the lipid interface.

Protein structure refinement based on paramagnetic NMR shifts: applications to wild-type and mutant forms of cytochrome c.

A new approach to NMR solution structure refinement is introduced that uses paramagnetic effects on nuclear chemical shifts as constraints in energy minimization or molecular dynamics calculations. Chemical shift differences between oxidized and reduced forms of horse cytochrome c for more than 300 protons were used as constraints to refine the structure of the wild-type protein in solution and to define the structural changes induced by a Leu 94 to Val mutation. A single round of constrained minimization, using the crystal structure as the starting point, converged to a low-energy structure with an RMS deviation between calculated and observed pseudo-contact shifts of 0.045 ppm, 7.5-fold lower than the starting structure. At the same time, the procedure provided stereospecific assignments for more than 45 pairs of methylene protons and methyl groups. Structural changes caused by the mutation were determined to a precision of better than 0.3 A. Structure determination based on dipolar paramagnetic (pseudocontact) shifts is applicable to molecules containing anisotropic paramagnetic centers with short electronic relaxation times, including numerous naturally occurring metalloproteins, as well as proteins or nucleic acids to which a paramagnetic metal ion or ligand may be attached. The long range of paramagnetic shift effects (up to 20 A from the iron in the case of cytochrome c) provides global structural constraints, which, in conjunction with conventional NMR distance and dihedral angle constraints, will enhance the precision of NMR solution structure determination.

Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.

Localized solution structure refinement of an F45W variant of ubiquitin using stochastic boundary molecular dynamics and NMR distance restraints.

The local structure within an 8-A radius around residue 45 of a recombinant F45W variant of human ubiquitin has been determined using 67 interproton distance restraints measured by two-dimensional proton NMR. Proton chemical shift evidence indicates that structural perturbations due to the F45W mutation are minimal and limited to the immediate vicinity of the site of mutation. Simulated annealing implemented with stochastic boundary molecular dynamics was applied to refine the structure of Trp 45 and 10 neighboring residues. The stochastic boundary method allowed the entire protein to be reassembled from the refined coordinates and the outlying unrefined coordinates with little distortion at the boundary. Refinement began with four low-energy indole ring orientations of F45W-substituted wild-type (WT) ubiquitin crystal coordinates. Distance restraints were derived from mostly long-range NOE cross peaks with 51 restraints involving the Trp 45 indole ring. Tandem refinements of 64 structures were done using either (1) upper and lower bounds derived from qualitative inspection of NOE crosspeak intensities or (2) quantitative analysis of cross-peak heights using the program MARDIGRAS. Though similar to those based on qualitative restraint, structures obtained using quantitative NOE analysis were superior in terms of precision and accuracy as measured by back-calculated sixth-root R factors. The six-membered portion of the indole ring is nearly coincident with the phenyl ring of the WT and the indole NH is exposed to solvent. Accommodation of the larger ring is accompanied by small perturbations in the backbone and a 120 degrees rotation of the chi 2 dihedral angle of Leu 50.

Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state.

Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.

Determinants of protein hydrogen exchange studied in equine cytochrome c.

The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 10(3) are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rates do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H-bonding contact with the H-exchange catalyst (OH(-)-ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H-bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 angstroms. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.

The evolutionarily conserved Dim1 protein defines a novel branch of the thioredoxin fold superfamily.

Dim1 is a small evolutionarily conserved protein essential for G2/M transition that has recently been implicated as a component of the mRNA splicing machinery. To date, the mechanism of Dim1 function remains poorly defined, in part because of the absence of informative sequence homologies between Dim1 and other functionally defined proteins or protein domains. We have used a combination of molecular modeling and NMR structural analysis to demonstrate that approximately 125 of the 142 amino acids of human Dim1 (hDim1) define a novel branch of the thioredoxin fold superfamily. Mutational analysis of Dim1 based on the predicted fold indicates that alterations in the region corresponding to the thioredoxin active site do not affect Dim1 activity. However, removal of a very short carboxy-terminal extension generates a dominant negative form of the protein [hDim1-(1-128)] that when overproduced induces cell cycle arrest in G2, via a mechanism likely to involve alteration of Dim1 association with partner molecules. In sum, this study identifies the Dim1 proteins as a novel sixth branch of the thioredoxin superfamily involved in cell cycle.

Activation of neuronal extracellular receptor kinase (ERK) in Alzheimer disease links oxidative stress to abnormal phosphorylation.

Responses to increased oxidative stress may be the common mechanism responsible for the varied cytopathology of Alzheimer disease (AD). A possible link in support of this hypothesis is that one of the most striking features of AD, the abnormal accumulation of highly phosphorylated tau and neurofilament proteins, may be brought about by extracellular receptor kinase (ERK) whose activation is a common response to oxidative stress. In this study, we demonstrate that activated ERK is specifically increased in the same vulnerable neurons in AD that are the site of oxidative damage and abnormal phosphorylation. These findings suggest that ERK dysregulation, likley resulting from oxidative stress, could play an important role in the increased phosphorylation of cytoskeletal proteins observed in AD.

Structure of the novel steroidal antibiotic squalamine determined by two-dimensional NMR spectroscopy.

Squalamine is a novel aminosterol recently isolated from the dogfish shark, Squalus acanthias. This water-soluble steroid exhibits potent antibacterial activity against both gram-negative and gram-positive bacteria. In addition, squalamine is fungicidal and induces osmotic lysis of protozoa. We report here the structural determination of squalamine, 3 beta-N-1-[N(3-[4-aminobutyl])-1,3 diaminopropane]-7 alpha,24 zeta-dihydroxy-5 alpha-cholestane 24-sulfate, which was deduced from the analysis of fast atom bombardment spectra and a series of two-dimensional nuclear magnetic resonance (NMR) spectra. Squalamine is a cationic steroid characterized by a condensation of an anionic bile salt intermediate with the polyamine, spermidine. This molecule is a potential host-defense agent in the shark, and provides insight into a new class of vertebrate antimicrobial molecules.

[Diagnostic value of angiodynography in pulsating space-occupying lesions]. / Diagnostische Wertigkeit der Angiodynographie bei pulsierenden Raumforderungen.

19 patients with undiagnosed pulsating masses occurring after angiography, by-pass surgery or trauma were examined by conventional ultrasound, duplex sonography, angiodynography and, in some cases, by angiography. There were nine pseudo-aneurysms and 13 haematomas; in each case the diagnosis was made by duplex sonography and by angiodynography. Pseudoaneurysms show the following typical signs on angiodynography: 1. Typical wave form during spectrum analysis, 2. Specific blood-flow pattern into and out of the aneurysm, 3. Turbulence within the aneurysm. Angiodynography provides a reliable, rapid and exact means of diagnosis. In future, this will be able to replace more invasive procedures such as angiography.

[Correspondence of mammographic and pathological-anatomical tumor extent in breast cancer]. / Ubereinstimmung von mammographischer und pathologisch-anatomischer Tumorausdehnung bei Mammakarzinom.

Pre-operative mammograms and exact information on the pathological-anatomical extent of the tumour were available in 198 patients with carcinoma of the breast. The tumour as seen on the mammogram was measured and compared with the histological information. Five of the 198 carcinomas (2 1/2%) could not be seen on the mammogram. In 69.7% of cases, mammography was suspicious of a carcinoma and in 27.8% a lesion was found that required further investigation. In 67.7%, the mammographic and pathological tumour extent agreed within 3 mm. In 18.2% the tumour appeared more than 3 mm greater, and in 9.6% more than 3 mm smaller than real size; the error was greater than 30 mm in only 3.2%.

Relation Between Wire Resistance and Fluid Pressure in the Transient Hot-Wire Method.

The resistance of metals is a function of applied pressure, and this dependence is large enough to be significant in the calibration of transient hot-wire thermal conductivity instruments. We recommend that for the highest possible accuracy, the instrument's hot wires should be calibrated in situ. If this is not possible, we recommend that a value of γ, the relative resistance change with pressure, of -2×10-5 MPa-1 be used to account for the pressure dependence of the platinum wire's resistance.

A High-Temperature Transient Hot-Wire Thermal Conductivity Apparatus for Fluids.

A new apparatus for measuring both the thermal conductivity and thermal diffusivity of fluids at temperatures from 220 to 775 K at pressures to 70 MPa is described. The instrument is based on the step-power-forced transient hot-wire technique. Two hot wires are arranged in different arms of a Wheatstone bridge such that the response of the shorter compensating wire is subtracted from the response of the primary wire. Both hot wires are 12.7 µm diameter platinum wire and are simultaneously used as electrical heat sources and as resistance thermometers. A microcomputer controls bridge nulling, applies the power pulse, monitors the bridge response, and stores the results. Performance of the instrument was verified with measurements on liquid toluene as well as argon and nitrogen gas. In particular, new data for the thermal conductivity of liquid toluene near the saturation line, between 298 and 550 K, are presented. These new data can be used to illustrate the importance of radiative heat transfer in transient hot-wire measurements. Thermal conductivity data for liquid toluene, which are corrected for radiation, are reported. The precision of the thermal conductivity data is ± 0.3% and the accuracy is about ±1%. The accuracy of the thermal diffusivity data is about ± 5%. From the measured thermal conductivity and thermal diffusivity, we can calculate the specific heat, Cp , of the fluid, provided that the density is measured, or available through an equation of state.

Absolute Steady-State Thermal Conductivity Measurements by Use of a Transient Hot-Wire System.

A transient hot-wire apparatus was used to measure the thermal conductivity of argon with both steady-state and transient methods. The effects of wire diameter, eccentricity of the wire in the cavity, axial conduction, and natural convection were accounted for in the analysis of the steady-state measurements. Based on measurements on argon, the relative uncertainty at the 95 % level of confidence of the new steady-state measurements is 2 % at low densities. Using the same hot wires, the relative uncertainty of the transient measurements is 1 % at the 95 % level of confidence. This is the first report of thermal conductivity measurements made by two different methods in the same apparatus. The steady-state method is shown to complement normal transient measurements at low densities, particularly for fluids where the thermophysical properties at low densities are not known with high accuracy.