A challenge to the evidence behind noise guidelines for UK hospitals.

BACKGROUND: Teams assessing hospital noise against international guidelines regularly find that noise exceeds perceived safe levels in clinical settings. The care of sick people may be inherently noisy but recent efforts to tackle the problem propose a wider scope to identify sources and qualities of noise as well as more precision with noise recording. AIMS: We sought to challenge the scientific evidence cited in the four major documents pertaining to hospital noise in the UK to clarify if evidence of harm from noise included in guidelines is available, contemporary and of high quality. METHODS: Our team of hearing-health clinicians, acoustic scientists and acoustic engineers have conducted a narrative scoping review focused on critically appraising four of the most cited guidelines against which noise is measured in healthcare settings in the UK. RESULTS: There is a lack of high-quality evidence for commonly accepted consequences of noise cited in current guidelines. CONCLUSIONS: The current evidence base for noise guidelines in a healthcare setting is largely based on subjective heterogeneous and inconclusive research. Whilst reduced noise is not disputed as potentially beneficial for patient care, further hypothesis-driven research and interventions assessing the benefits or outcomes of any such intervention should be sought to produce high-quality evidence of relevance on the clinical coalface.

Structure of the nucleotide activation switch in glycogen phosphorylase a.

Adenosine monophosphate is required for the activation of glycogen phosphorylase b and for release of the inhibition of phosphorylase a by glucose. Two molecules of adenosine monophosphate (AMP) bind to symmetry related sites at the subunit interface of the phosphorylase dimer. Adenosine triphosphate (ATP) binds to the same site, but does not promote catalytic activity. The structure of glucose-inhibited phosphorylase a bound to AMP and also of the complex formed with glucose and ATP is described. Crystallographic refinement of these complexes reveals that structural changes are associated with AMP but not ATP binding. The origin of these effects can be traced to different effector binding modes exhibited by AMP and ATP, respectively. The conformational changes associated with AMP binding traverse multiple paths in the enzyme and link the effector and catalytic sites.

Domain separation in the activation of glycogen phosphorylase a.

The crystal structure of glycogen phosphorylase a complexed with its substrates, orthophosphate and maltopentaose, has been determined and refined at a resolution of 2.8 angstroms. With oligosaccaride bound at the glycogen storage site, the phosphate ion binds at the catalytic site and causes the regulatory and catalytic domains to separate with the loss of stabilizing interactions between them. Homotropic cooperativity between the active sites of the allosteric dimer results from rearrangements in isologous contacts between symmetry-related helices in the subunit interface. The conformational changes in the core of the interface are correlated with those observed on covalent activation by phosphorylation at Ser14 (phosphorylase b----a).

Converting tissue plasminogen activator to a zymogen: a regulatory triad of Asp-His-Ser.

Unlike most serine proteases of the chymotrypsin family, tissue-type plasminogen activator (tPA) is secreted from cells as an active, single-chain enzyme with a catalytic efficiency only slightly lower than that of the proteolytically cleaved form. A zymogenic mutant of tPA has been engineered that displays a reduction in catalytic efficiency by a factor of 141 in the single-chain form while retaining full activity in the cleaved form. The residues introduced in the mutant, serine 292 and histidine 305, are proposed to form a hydrogen-bonded network with aspartate 477, similar to the aspartate 194-histidine 40-serine 32 network found to stabilize the zymogen chymotrypsinogen.

Structural basis for the activation of glycogen phosphorylase b by adenosine monophosphate.

The three-dimensional structure of the activated state of glycogen phosphorylase (GP) as induced by adenosine monophosphate (AMP) has been determined from crystals of pyridoxalpyrophosphoryl-GP. The same quaternary changes relative to the inactive conformation as those induced by phosphorylation are induced by AMP, although the two regulatory signals function through different local structural mechanisms. Moreover, previous descriptions of the phosphorylase active state have been extended by demonstrating that, on activation, the amino- and carboxyl-terminal domains of GP rotate apart by 5 degrees, thereby increasing access of substrates to the catalytic site. The structure also reveals previously unobserved interactions with the nucleotide that accounts for the specificity of the nucleotide binding site for AMP in preference to inosine monophosphate.

Dimerization in MAP-kinase signaling.

The stimulus-dependent nuclear localization of the extracellular-signal- regulated kinases ERK1 and ERK2 is required for many of their actions, including induction of neurites in PC12 cells and transformation of fibroblasts. Phosphorylation of ERK2 causes it to form dimers, and the most flexible portions of the ERK2 molecule provide the surfaces for dimerization. It is thought that dimerization promotes nuclear localization of ERK2 by its effects on import, export or retention in cytoplasmic and nuclear compartments. Dimerization might also influence substrate interactions.

A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation.

BACKGROUND: Mitogen-activated protein (MAP) kinases are ubiquitous components of many signal transduction pathways. Constitutively active variants have been isolated for every component of the extracellular-signal-regulated kinase 1 (ERK1) and ERK2 MAP kinase pathway except for the ERK itself. RESULTS: To create an activated ERK2 variant, we fused ERK2 to the low activity form of its upstream regulator, the MAP kinase kinase MEK1. The ERK2 in this fusion protein was active in the absence of extracellular signals. Expression of the fusion protein in mammalian cells did not activate endogenous ERK1 or ERK2. It was sufficient, however, to induce activation of the transcription factors Elk-1 and AP-1, neurite extension in PC12 cells in the absence of nerve growth factor, and foci of morphologically and growth-transformed NIH3T3 cells, if the fusion protein was localized to the nucleus. A cytoplasmic fusion protein was without effect. CONCLUSIONS: Activation of ERK2 is sufficient to cause several transcriptional and phenotypic responses in mammalian cells. Nuclear localization of activated ERK2 is required to induce these events.

Protein kinases.

The structures of four serine/threonine protein kinases have been determined recently. By comparing these structures with that of the cAMP-dependent protein kinase (cAPK), it is now possible to see how the activity of these regulatory enzymes is controlled. Low activity is maintained through the conformation of the phosphorylation lip, domain rotations, and binding of substrate analog inhibitors and autoinhibitory domains.

Serpins and other covalent protease inhibitors.

Serpins are irreversible covalent 'suicide' protease inhibitors. In the past two years, important advances in the structural biology of serpins have been forthcoming with the crystal structures of a covalent complex between trypsin and alpha1-antitrypsin, and of a Michaelis encounter complex between trypsin S195A and serpin 1B from Manduca sexta. These structures have helped elucidate many aspects of the mechanism of action of serpins. Also, the crystal structure of the cysteine protease caspase-8 in complex with the inhibitor p35 has revealed a new family of suicide protease inhibitors.

Estrogen status and skeletal muscle recovery from disuse atrophy.

Although estrogen loss can alter skeletal muscle recovery from disuse, the specific components of muscle regrowth that are estrogen sensitive have not been described. The primary purpose of this study was to determine the components of skeletal muscle mass recovery that are biological targets of estrogen. Intact, ovariectomized (OVX), and ovariectomized with 17beta-estradiol replacement (OVX+E2) female rats were subjected to hindlimb suspension for 10 days and then returned to normal cage ambulation for the duration of recovery. Soleus muscle mass returned to control levels by day 7 of recovery in the intact animals, whereas OVX soleus mass did not recover until day 14. Intact rats recovered soleus mean myofiber cross-sectional area (CSA) by day 14 of recovery, whereas the OVX soleus remained decreased (42%) at day 14. OVX mean fiber CSA did return to control levels by day 28 of recovery. The OVX+E2 treatment group recovered mean CSA at day 14, as in the intact animals. Myofibers demonstrating central nuclei were increased at day 14 in the OVX group, but not in intact or OVX+E2 animals. The percent noncontractile tissue was also increased 29% in OVX muscle at day 14, but not in either intact or OVX+E2 groups. In addition, collagen 1a mRNA was increased 45% in OVX muscle at day 14 of recovery. These results suggest that myofiber growth, myofiber regeneration, and extracellular matrix remodeling are estrogen-sensitive components of soleus muscle mass recovery from disuse atrophy.

Serpins: the uncut version.

The structure of active antithrombin, the first active serpin to be solved, sheds new light on the conformational forms of this important class of inhibitor.

Activity of the MAP kinase ERK2 is controlled by a flexible surface loop.

BACKGROUND: The mitogen-activated protein (MAP) kinase, ERK2, is a tightly regulated enzyme in the ubiquitous Ras-activated protein kinase cascade. ERK2 is activated by phosphorylation at two sites, Y185 and T183, that lie in the phosphorylation lip at the mouth of the catalytic site. To ascertain the role of these two residues in securing the low-activity conformation of the enzymes we have carried out crystallographic analyses and assays of phosphorylation-site mutants of ERK2. RESULTS: The crystal structures of four mutants, T183E (threonine at residue 183 is replaced by glutamate), Y185E, Y185F and the double mutant T183E/Y185E, were determined. When T183 is replaced by glutamate, few conformational changes are observed. By contrast, when Y185 is replaced by glutamate, 19 residues become disordered, including the entire phosphorylation lip and an adjacent loop. The conservative substitution of phenylalanine for Y185 also induces relatively large conformational changes. A binding site for phosphotyrosine in the active enzyme is putatively identified on the basis of the high-resolution refinement of the structure of wild-type ERK2. CONCLUSIONS: The remarkable disorder observed throughout the phosphorylation lip when Y185 is mutated shows that the stability of the phosphorylation lip is rather low. Therefore, only modest amounts of binding energy will be required to dislodge the lip for phosphorylation, and it is likely that these residues will be involved in conformational changes associated with both with binding to kinases and phosphatases and with activation. Furthermore, the low-activity structure is specifically dependent on Y185, whereas there is no such dependency on T183. Both residues, however, participate in forming the active enzyme, contributing to its tight control.

The structure of active serpin 1K from Manduca sexta.

BACKGROUND: The reactive center loops (RCL) of serpins undergo large conformational changes triggered by the interaction with their target protease. Available crystallographic data suggest that the serpin RCL is polymorphic, but the relevance of the observed conformations to the competent active structure and the conformational changes that occur on binding target protease has remained obscure. New high-resolution data on an active serpin, serpin 1K from the moth hornworm Manduca sexta, provide insights into how active serpins are stabilized and how conformational changes are induced by protease binding. RESULTS: The 2.1 A structure shows that the RCL of serpin 1K, like that of active alpha1-antitrypsin, is canonical, complimentary and ready to bind to the target protease between P3 and P3 (where P refers to standard protease nomenclature),. In the hinge region (P17-P13), however, the RCL of serpin 1K, like ovalbumin and alpha1-antichymotrypsin, forms tight interactions that stabilize the five-stranded closed form of betasheet A. These interactions are not present in, and are not compatible with, the observed structure of active alpha1-antitrypsin. CONCLUSIONS: Serpin 1K may represent the best resting conformation for serpins - canonical near P1, but stabilized in the closed conformation of betasheet A. By comparison with other active serpins, especially alpha1-antitrypsin, a model is proposed in which interaction with the target protease near P1 leads to conformational changes in betasheet A of the serpin.

Structural basis of inhibitor selectivity in MAP kinases.

BACKGROUND: The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear. RESULTS: We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine. CONCLUSIONS: The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.

Left ventricular hypertrophy in ascending aortic stenosis mice: anoikis and the progression to early failure.

BACKGROUND: To determine potential mechanisms of the transition from hypertrophy to very early failure, we examined apoptosis in a model of ascending aortic stenosis (AS) in male FVB/n mice. METHODS AND RESULTS: Compared with age-matched controls, 4-week and 7-week AS animals (n=12 to 16 per group) had increased ratios of left ventricular weight to body weight (4.7+/-0.7 versus 3.1+/-0.2 and 5. 7+/-0.4 versus 2.7+/-0.1 mg/g, respectively, P<0.05) with similar body weights. Myocyte width was also increased in 4-week and 7-week AS mice compared with controls (19.0+/-0.8 and 25.2+/-1.8 versus 14. 1+/-0.5 microm, respectively, P<0.01). By 7 weeks, AS myocytes displayed branching with distinct differences in intercalated disk size and staining for beta(1)-integrin on both cell surface and adjacent extracellular matrix. In vivo left ventricular systolic developed pressure per gram as well as endocardial fractional shortening were similar in 4-week AS and controls but depressed in 7-week AS mice. Myocyte apoptosis estimated by in situ nick end-labeling (TUNEL) was extremely rare in 4-week AS and control mice; however, a low prevalence of TUNEL-positive myocytes and DNA laddering were detected in 7-week AS mice. The specificity of TUNEL labeling was confirmed by in situ ligation of hairpin oligonucleotides. CONCLUSIONS: Our findings indicate that myocyte apoptosis develops during the transition from hypertrophy to early failure in mice with chronic biomechanical stress and support the hypothesis that the disruption of normal myocyte anchorage to adjacent extracellular matrix and cells, a process called anoikis, may signal apoptosis.

Crystallization and preliminary X-ray studies of extracellular signal-regulated kinase-2/MAP kinase with an incorporated His-tag.

The extracellular signal-regulated kinase ERK2, a member of the protein kinase superfamily, phosphorylates a variety of cellular proteins in response to extracellular signals. ERK2 expressed in Escherichia coli as a fusion protein with the sequence Ala-His6 at the N terminus has low basal activity and very low levels of phosphate incorporation, but can be fully activated. The Ala-His6 ERK2 as expressed in the unphosphorylated form has been crystallized in space group P2(1). The cell constants are a = 49.32 A, b = 71.42 A, c = 61.25 A, and beta = 109.75 degrees, and the crystals diffract to better than 1.8 A resolution.

Crystallization and preliminary X-ray data for the A-isozyme of O-acetylserine sulfhydrylase from Salmonella typhimurium.

The A-isozyme of O-acetylserine sulfhydrylase, a pyridoxal phosphate-dependent enzyme isolated from Salmonella typhimurium catalyzes the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The pyridoxal form of the enzyme has been crystallized in two different forms. One form is in the orthorhombic space group P2(1)2(1)2(1) with cell constants a = 144.4 A, b = 96.9 A and c = 54.3 A and contains two monomers each of molecular weight 34,000 per asymmetric unit. The second form is in a hexagonal space group with unit cell dimensions a = b = 115 A, and c = 348 A and contains two 68,000 dimers per asymmetric unit. Complete native enzyme data sets have been collected for both crystal forms using an R-Axis II detector. A search for suitable heavy-atom derivatives is underway. Although both crystal forms diffract X-rays to better than 2.5 A, the orthorhombic form is more suited to a detailed structural analysis due to the extended lifetime in the X-ray beam and the relative size of the unit cell.

Long inverted repeats are an at-risk motif for recombination in mammalian cells.

Certain DNA sequence motifs and structures can promote genomic instability. We have explored instability induced in mouse cells by long inverted repeats (LIRs). A cassette was constructed containing a herpes simplex virus thymidine kinase (tk) gene into which was inserted an LIR composed of two inverted copies of a 1.1-kb yeast URA3 gene sequence separated by a 200-bp spacer sequence. The tk gene was introduced into the genome of mouse Ltk(-) fibroblasts either by itself or in conjunction with a closely linked tk gene that was disrupted by an 8-bp XhoI linker insertion; rates of intrachromosomal homologous recombination between the markers were determined. Recombination between the two tk alleles was stimulated 5-fold by the LIR, as compared to a long direct repeat (LDR) insert, resulting in nearly 10(-5) events per cell per generation. Of the tk(+) segregants recovered from LIR-containing cell lines, 14% arose from gene conversions that eliminated the LIR, as compared to 3% of the tk(+) segregants from LDR cell lines, corresponding to a >20-fold increase in deletions at the LIR hotspot. Thus, an LIR, which is a common motif in mammalian genomes, is at risk for the stimulation of homologous recombination and possibly other genetic rearrangements.

Relative dependence of different outputs of the Saccharomyces cerevisiae pheromone response pathway on the MAP kinase Fus3p.

Fus3p and Kss1p act at the end of a conserved signaling cascade that mediates numerous cellular responses for mating. To determine the role of Fus3p in different outputs, we isolated and characterized a series of partial-function fus3 point mutants for their ability to phosphorylate a substrate (Ste7p), activate Ste12p, undergo G1 arrest, form shmoos, select partners, mate, and recover. All the mutations lie in residues that are conserved among MAP kinases and are predicted to affect either enzyme activity or binding to Ste7p or substrates. The data argue that Fus3p regulates the various outputs assayed through the phosphorylation of multiple substrates. Different levels of Fus3p function are required for individual outputs, with the most function required for shmoo formation, the terminal output. The ability of Fus3p to promote shmoo formation strongly correlates with its ability to promote G1 arrest, suggesting that the two events are coupled. Fus3p promotes recovery through a mechanism that is distinct from its ability to promote G1 arrest and may involve a mechanism that does not require kinase activity. Moreover, catalytically inactive Fus3p inhibits the ability of active Fus3p to activate Ste12p and hastens recovery without blocking G1 arrest or shmoo formation. These results raise the possibility that in the absence of sustained activation of Fus3p, catalytically inactive Fus3p blocks further differentiation by restoring mitotic growth. Finally, suppression analysis argues that Kss1p contributes to the overall pheromone response in a wild-type strain, but that Fus3p is the critical kinase for all of the outputs tested.

Modeling of the spatial structure of eukaryotic ornithine decarboxylases.

We used sequence and structural comparisons to determine the fold for eukaryotic ornithine decarboxylase, which we found is related to alanine racemase. These enzymes have no detectable sequence identity with any protein of known structure, including three pyridoxal phosphate-utilizing enzymes. Our studies suggest that the N-terminal domain of ornithine decarboxylase folds into a beta/alpha-barrel. Through the analysis of known barrel structures we developed a topographic model of the pyridoxal phosphate-binding domain of ornithine decarboxylase, which predicts that the Schiff base lysine and a conserved glycine-rich sequence both map to the C-termini of the beta-strands. Other residues in this domain that are likely to have essential roles in catalysis, substrate, and cofactor binding were also identified, suggesting that this model will be a suitable guide to mutagenic analysis of the enzyme mechanism.