Comparison of different intubation techniques performed inside a moving ambulance: a manikin study.

OBJECTIVE. Airway management and endotracheal intubation may be required urgently when a patient deteriorates in an ambulance or aircraft during interhospital transfer or in a prehospital setting. The objectives of this study were: (1) to compare the effectiveness of conventional intubation by Macintosh laryngoscope in a moving ambulance versus that in a static ambulance; and (2) to compare the effectiveness of inverse intubation and GlideScope laryngoscopy with conventional intubation inside a moving ambulance. DESIGN. Comparative experimental study. SETTING. The experiment was conducted in an ambulance provided by the Auxiliary Medical Service in Hong Kong. PARTICIPANTS. A group of 22 doctors performed endotracheal intubation on manikins with Macintosh laryngoscope in a static and moving ambulance. In addition, they performed conventional Macintosh intubation, inverse intubation with Macintosh laryngoscope, and GlideScope intubation in a moving ambulance in both normal and simulated difficult airways. MAIN OUTCOME MEASURES. The primary outcome was the rate of successful intubation. The secondary outcomes were time taken for intubation, subjective glottis visualisation grading, and eventful intubation (oesophageal intubation, intubation time >60 seconds, and incisor breakage) with different techniques or devices. RESULTS. In normal airways, conventional Macintosh intubation in a static ambulance (95.5%), conventional intubation in a moving ambulance (95.5%), as well as GlideScope intubation in a moving ambulance (95.5%) were associated with high success rates; the success rate of inverse intubation was comparatively low (54.5%; P=0.004). In difficult airways, conventional Macintosh intubation in a static ambulance (86.4%), conventional intubation in a moving ambulance (90.9%), and GlideScope intubation in a moving ambulance (100%) were associated with high success rates; the success rate of inverse intubation was comparatively lower (40.9%; P=0.034). CONCLUSIONS. En-route intubation in an ambulance by conventional Macintosh laryngoscopy is superior to inverse intubation unless the cephalad access is impossible. GlideScope laryngoscopy appears to be associated with lower rates of eventful intubation in difficult airways and has better laryngoscopic view versus inverse intubation.

Collapsin response mediator protein-1 (CRMP1) acts as an invasion and metastasis suppressor of prostate cancer via its suppression of epithelial-mesenchymal transition and remodeling of actin cytoskeleton organization.

The cancer cells can acquire migration and invasion capacities during the metastasis process through the developmental regulatory program epithelial-mesenchymal-transition (EMT), and through its reverse process mesenchymal-epithelial transition cancer cells can recolonize at distant metastatic sites. Among the multifaceted effects exerted by this program, reorganization of actin cytoskeleton is the key mechanical drive for the invasive properties gained by cancer cells. Collapsin response mediator protein-1 (CRMP1) is a cytosolic phosphoprotein and originally characterized as the mediator of semaphorin 3A signaling involved in axon differentiation during neural development. Here we report that CRMP1 can act as a suppressor of tumorigenicity and metastasis in prostate cancer cells. We demonstrated that CRMP1 exhibited a decreased expression pattern in high-grade prostate cancer tissues and many prostate cancer cell lines, and its downregulation in cancer cells was attributed to histone deacetylation and direct repression of its gene by the EMT regulator Snail. Functional analyses revealed that CRMP1 suppressed EMT in prostate cancer cells, as its knockdown could trigger EMT and enhance in vitro invasion capacity, whereas its overexpression could inhibit EMT and suppress both in vitro invasion and in vivo metastasis capacities of prostate cancer cells. Moreover, CRMP1 overexpression could significantly confer resistance to EMT induced by Snail or transforming growth factor-ß1 in prostatic epithelial cells and prostate cancer cells. Finally, we demonstrated that CRMP1 could associate with actin and WAVE1, an activator of actin nucleation complex Arp2/3, and also its knockdown could stabilize F-actin and trigger the formation of stress fibers in prostate cancer cells. Together, our study shows that CRMP1 acts an EMT and metastasis suppressor in prostate cancer cells via its regulation of actin polymerization and also suggests that targeting the CRMP1-actin signaling in actin organization could be a potential strategy for management of prostate cancer metastasis.

Expression and functional study of estrogen receptor-related receptors in human prostatic cells and tissues.

Estrogen receptor-related receptors (ERRs; alpha, beta, gamma) are orphan nuclear receptors and constitutively active without binding to estrogen. Like estrogen receptors (ERs), ERRs bind to estrogen receptor elements and estrogen receptor element-related repeats. Growing evidence suggests that ERRs can cross-talk with ERs in different cell types via competition for DNA sites and coactivators. We hypothesize that ERRs might play regulatory roles in normal and neoplastic prostatic cells by sharing similar ER-mediated pathways or acting independently. In this study, we investigated mRNA and protein expression patterns of three ERR members in normal human prostate epithelial cells, established cell lines, cancer xenografts, and prostatic tissues. Additionally, effects of transient transfection of ERRs on prostatic cell proliferation and ER expression were also examined. RT-PCR showed that ERRalpha and ERRgamma transcripts were detected in most cell lines and xenografts, whereas ERRbeta was detected in normal epithelial cells and few immortalized cell lines but not in most cancer lines. Similar results were demonstrated in clinical prostatic specimens. Western blottings and immunohistochemistry confirmed similar expression patterns that ERR proteins were detected as nuclear proteins in epithelial cells, whereas their expressions became reduced or undetected in neoplastic prostatic cells. Transient transfection confirmed that ERRs were expressed in prostatic cells as nuclear proteins and transcriptionally active in the absence of estradiol. Transfection results showed that overexpression of ERRs inhibited cell proliferation and repressed ERalpha transcription in PC-3 cells. Our study shows that ERRs, which are coexpressed with ERs in prostatic cells, could regulate cell growth and modulate ER-mediated pathways via interference on ERalpha transcription in prostatic cells.

Cold denaturation of barstar: 1H, 15N and 13C NMR assignment and characterisation of residual structure.

Detection of residual structure in denatured proteins is of interest because fleetingly structured regions may be initiation points of the folding pathway. Residual structure in this context is not the definition of one stable conformation but a population phenomenon. Acid, thermal and solvent-denatured states have recently been examined by NMR spectroscopy, but cold-denatured states have not been characterised to date. Cold denaturation is a general phenomenon of globular proteins, which provides a convenient route for studying early events in protein folding: such states can be induced to fold and be monitored on a submillisecond time scale by temperature-jump methods. Here, we use NMR spectroscopy to define residual structure in cold-denatured barstar. The cold-denatured state becomes significantly populated in the presence of increasing concentrations of urea and lower temperature. In the presence of 3 M urea, the double mutant of barstar in which Cys40 and Cys82 are both mutated to Ala (C40/82A) is completely and reversibly denatured at 278 K, a temperature that is accessible to NMR experiments. This cold-denatured state of barstar was assigned by heteronuclear NMR experiments and structural parameters such as NOE, coupling constants and chemical shifts were derived. Based on the excellent dispersion in a HSQC-NOESY-HSQC experiment, dNN(i,i+1) NOEs were observed for almost all residues. This allowed us to normalise NOE intensities as the NOE: diagonal ratio dNN(i,i+1) NOE (sigma NN) and the NOE ratio of d(alpha N(i+1,i+1)):d(alpha N(i,i+1)) (sigma N alpha/sigma alpha N). This approach reveals residual structure populating the alpha-region of the (phi, psi) conformational space in the regions corresponding to the first and the second helices and near the end of the second beta-strand of native barstar, whereas the C-terminal region that corresponds to the fourth helix and the third beta-strand is in a random coil conformation. The results suggest that the first and the second helices are potential initiation sites for the folding of barstar. The details presented here provide the starting point for the study of rapid folding of cold-denatured barstar.

NMR 15N relaxation and structural studies reveal slow conformational exchange in barstar C40/82A.

Barstar an 89-residue protein consisting of four helices and a three-stranded parallel beta-sheet, is the intracellular inhibitor of the endoribonuclease barnase. Barstar C40/82A, a mutant in which the two cysteine residues have been replaced by alanine, has been used as a pseudo wild-type in folding studies and in the crystal structure of the barnase:barstar C40/82A complex. We have determined a high resolution solution structure of barstar C40/82A. The structures of barstar C40/82A and the wild-type are superimposable. A comparison with the crystal structure of the barnase:barstar C40/82A complex revealed subtle differences in the regions involved in the binding of barstar to barnase. Side-chain rotations of residues Asn33, Asp35 and Asp39 and a movement of the binding loop (Pro27-Glu32) towards the binding site of barnase facilitate the formation of interface hydrogen bonds and aromatic contacts in the complex. Extreme line broadening and missing signals in 1H-15N correlation spectra indicate substantial conformational exchange for a large subset of residues. 15N relaxation data at two magnetic field strengths, 11.74 T and 14.10 T, were used to estimate exchange contributions and to map the spectral density function at five frequencies: 0, 50, 60, 450 and 540 MHz. Based on these results, model-free calculations with the inclusion of estimated exchange contributions were used to derive order parameters and internal correlation times. The validity of this approach has been investigated with model-free calculations that incorporate longitudinal relaxation rates and heteronuclear 1H-15N NOE data only at 11.74 T and 14.10 T. The relaxation data suggest substantial conformational exchange in regions of barstar C40/82A, including the binding loop, the second and the third helices, and the second and the third strands. Amide proton exchange experiments suggest a stable hydrogen bond network for all helices and sheets except the third helix and the C-terminal of the second and the third strands. The combined results indicate a rigid body movement of the second helix and twisting motions of the beta-sheet of barstar, which might be important for the interaction with barnase.

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

Molecular cloning and functional study of rat estrogen receptor-related receptor gamma in rat prostatic cells.

BACKGROUND: Based on high homology of ERRs with ERs, we hypothesize that ERRs might functionally cross talk with ERs or independently in prostatic cells. METHODS: We examined the ERRgamma expressions in rat prostates and Nb rat prostate cancer model, and its growth regulation in stable transfectants of prostatic cells. RESULTS: We cloned the ERRgamma cDNA from rat prostate by RACE-PCR. Its expression was confirmed by Northern and immunoblottings. Real-time RT-PCR showed that its expression in castrated prostates was androgen-dependent. ERRgamma was expressed in prostatic epithelial cells, but showed reduced expressions in neoplastic prostates. Transfections confirmed that ERRgamma was expressed in prostatic cells as nuclear protein and transcriptionally active without estradiol. Its overexpression in ERRgamma-stable transfectants of NbE-1 and MAT-Lu cells inhibited their in vitro proliferation, anchorage-independent growth in soft-agar and tumorigenicity in nude mice. CONCLUSIONS: Our studies show that ERRgamma is functionally expressed in rat prostate and may play anti-proliferative actions in prostatic cells. Its co-expression with ERs suggests that besides ERs, ligand-independent ERRgamma is also involved in prostatic growth and functions.

Barstar has a highly dynamic hydrophobic core: evidence from molecular dynamics simulations and nuclear magnetic resonance relaxation data.

The dynamic behavior of the ribonuclease inhibitor barstar has been investigated by molecular dynamics (MD) simulations in explicit water. Two 2.5 ns MD simulations were performed, and an ensemble of 25 000 structures was generated. This ensemble reproduces the solution structures and is consistent with the experimental structural restraints from NMR spectroscopy. Reorientation of the backbone NH bond vectors and side chain methyl groups was monitored by calculation of autocorrelation functions and the generalized S2 order parameters. Order parameters derived for motion in the approximately 100 ps time scale were compared with those obtained from NMR relaxation measurements. Consistent with experiment, the backbone NH bond vectors were relatively rigid. In contrast, the side chain methyl groups exhibited a wide dynamic range, from restricted motion comparable to that of the backbone to rapid unrestricted motion. The order parameters for the methyl groups correlate well with their spatial separation from the backbone and are residue-type dependent. Smaller S2axis values were observed for leucine methyl groups, in part due to side chain hopping between two predominant rotamers (g+t and tg-). Motions such as the flipping of aromatic rings and the hopping of leucine side chains were prevalent within the hydrophobic core, suggesting that the core is fluid-like with low energy barriers between native conformational substates. Thus, our studies suggest that the entropy of the native state can be significant and should not be discounted in thermodynamic considerations of protein folding. On the basis of our results, the side chain motion represents the primary source of the residual entropy of the native state and entropic considerations based solely on backbone dynamics would be incomplete.

Initiation sites of protein folding by NMR analysis.

Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable.

Mechanism of rescue of common p53 cancer mutations by second-site suppressor mutations.

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA-binding activity of such mutants to understand the mechanism of second-site suppressor mutations. Double-mutant cycles show that N239Y and N268D act as 'global stability' suppressors by increasing the stability of the cancer mutants G245S and V143A-the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.

Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: description of the folding pathway.

Residual structure in the denatured state of a protein may contain clues about the early events in folding. We have simulated by molecular dynamics the denatured state of barnase, which has been studied by NMR spectroscopy. An ensemble of 10(4) structures was generated after 2 ns of unfolding and following for a further 2 ns. The ensemble was heterogeneous, but there was nonrandom, residual structure with persistent interactions. Helical structure in the C-terminal portion of helix alpha1 (residues 13-17) and in helix alpha2 as well as a turn and nonnative hydrophobic clustering between beta3 and beta4 were observed, consistent with NMR data. In addition, there were tertiary contacts between residues in alpha1 and the C-terminal portion of the beta-sheet. The simulated structures allow the rudimentary NMR data to be fleshed out. The consistency between simulation and experiment inspires confidence in the methods. A description of the folding pathway of barnase from the denatured to the native state can be constructed by combining the simulation with experimental data from phi value analysis and NMR.

Crystallization and preliminary crystallographic studies of a ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer.

Ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer is a good model for the study of the thermostability of proteins. It has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as precipitant at 290 K. The crystal belongs to the hexagonal space group P6(1)/P6(5), with unit-cell parameters a = b = 48.32, c = 86.42 A. The asymmetric unit contains a single molecule of L30e, with a corresponding crystal volume per protein mass (V(M)) of 2.68 A(3) Da(-1) and a solvent content of 54%. A complete data set diffracting to 1.96 A resolution was collected from a single crystal at 100 K.

Hot-spot mutants of p53 core domain evince characteristic local structural changes.

Most of the oncogenic mutations in the tumor suppressor p53 map to its DNA-binding (core) domain. It is thus a potential target in cancer therapy for rescue by drugs. To begin to understand how mutation inactivates p53 and hence to provide a structural basis for drug design, we have compared structures of wild-type and mutant p53 core domains in solution by NMR spectroscopy. Structural changes introduced by five hot-spot mutations (V143A, G245S, R248Q, R249S, and R273H) were monitored by chemical-shift changes. Only localized changes are observed for G245S, R248Q, R249S, and R273H, suggesting that the overall tertiary folds of these mutant proteins are similar to that of wild type. Structural changes in R273H are found mainly in the loop-sheet-helix motif and the loop L3 of the core domain. Mutations in L3 (G245S, R248Q, and R249S) introduce structural changes in the loop L2 and L3 as well as terminal residues of strands 4, 9, and 10. It is noteworthy that R248Q, which is often regarded as a contact mutant that affects only interactions with DNA, introduces structural changes as extensive as the other loop L3 mutations (G245S and R249S). These changes suggest that R248Q is also a structural mutant that perturbs the structure of loop L2-L3 regions of the p53 core domain. In contrast to other mutants, replacement of the core residue valine 143 to alanine causes chemical-shift changes in almost all residues in the beta-sandwich and the DNA-binding surface. Long-range effects of V143A mutation may affect the specificity of DNA binding.

Protein folding from a highly disordered denatured state: the folding pathway of chymotrypsin inhibitor 2 at atomic resolution.

Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation-condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution.

Engineering of a mini-trichosanthin that has lower antigenicity by deleting its C-terminal amino acid residues.

Trichosanthin is a ribosome-inactivating protein that possesses antitumor and antiviral activities. Clinical trials of trichosanthin on AIDS patients, however, elicit anaphylactic reactions. To reduce the antigenicity of trichosanthin as a drug while preserving its biological activity, the C-terminal domain (residues 203 to 247), which contains a putative antigenic site, was systemically deleted. We have found that the minimum length of trichosanthin that can fold into an active conformation is residue 1 to 240. The mini-trichosanthin (C7) generated by deleting the last seven C-terminal amino acid residues has 2.7-fold decrease in antigenicity, 10-fold reduction in in vitro ribosome-inactivation activity, and in vivo cytotoxicity toward K562 cells, and 2-fold reduction in abortificient activity. Structural analyses of C7 indicate decrease in the helix content, increased exposure of Trp192, and lower thermodynamic stability. The deletion of the C-terminal residues (Leu241 to Ala247) probably perturbs local structure of the C-terminal antigenic epitope that results in the decrease in antigenicity and activities of C7.

Structure/function relationship study of Gln156, Glu160 and Glu189 in the active site of trichosanthin.

Trichosanthin is a protein used medicinally in China for abortifacient purposes. It is also an RNA N-glycosidase which inactivates eukaryotic ribosomes by removing adenine4324 from 28S rRNA. Site-directed mutagenesis was performed to probe the role of Gln156, Glu160 and Glu189 in the active site of trichosanthin. The purified altered proteins were assayed for their potency in inhibiting in vitro protein synthesis. The data indicate Glu160 is involved in the catalytic reaction. Kinetics studies suggest the carboxylate group of Glu160 serves to stabilize the transition-state complex. Similar to ricin A, the variant [E160A]trichosanthin is more potent than [E160D]trichosanthin. This is because Glu189 serves as a back-up of the carboxylate group in case Glu160 is mutated to alanine. However, removal of Glu189 in the presence of Glu160 does not affect the ID50 value drastically. An activity of 1800-fold less than that of the wild-type protein was found when both Glu160 and Glu189 were changed to alanine, indicating that some other residues in the active site are also taken part in the lowering of energy barrier for the catalytic reaction. Although Gln156 is highly conserved in related proteins, its mutation to alanine only slightly decreases the activity, showing that this residue does not participate directly in catalysis.