The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand binding studies reveal the location of the active site.

Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes. By applying a combination of residue-specific isotope labeling and protein deuteration strategies tailored to yield specific information, we were able to determine the solution structure and study structure-activity relationships of 3,4-dihydroxy-2-butanone-4-phosphate synthase, a 47-kDa enzyme from the Escherichia coli riboflavin biosynthesis pathway and an attractive target for novel antibiotics. Our investigations of the enzyme's ligand binding by NMR and site-directed mutagenesis yields a conclusive picture of the location and identity of residues directly involved in substrate binding and catalysis. Our studies illustrate the power of state-of-the-art NMR techniques for the structural characterization and investigation of ligand binding in protein complexes approaching the 50-kDa range in solution.

A protocol for identification of early bulbar signs in amyotrophic lateral sclerosis.

The purpose of this project is to identify characteristics that may be of assistance in establishing the diagnosis and monitoring early progression of bulbar dysfunction in patients with Amyotrophic Lateral Sclerosis (ALS). Early identification of bulbar dysfunction would assist in clinical trials and management decisions. A database of 218 clinic visits of patients with ALS was developed and formed the basis for these analyses. As a framework for the description of our methodology, the Disablement Model [World Health Organization. WHO International classification of impairment, activity, and participation: beginner's guide. In: WHO, editor. Beta-1 draft for field trials; 1999] was utilized. Our data identified that the strongest early predictors of bulbar speech dysfunction include altered voice quality (laryngeal control), speaking rate, and communication effectiveness. A protocol for measuring these speech parameters was therefore undertaken. This paper presents the protocol used to measure these bulbar parameters.

Development of a test of physical performance for the nursing home setting.

PURPOSE: This study was undertaken to develop a performance-based instrument to measure a range of function present among nursing home (NH) residents and to establish the reliability and validity of the measures. DESIGN AND METHODS: Fourteen items integral to daily life in a NH setting were administered to 95 NH residents with sufficient cognitive ability to follow a one-step command at baseline, 1 week later, and 6 months later. Intraclass correlation coefficients and Cronbach's coefficient alpha were calculated for reliability estimates. The Minimum Data Set (MDS), Katz, and Multidimensional Observational Scale for Elderly Subjects (MOSES) were used to establish concurrent validity. Factor analysis, correlation matrices, and other objective criteria were used for item reduction. RESULTS: Test-retest reliability for items ranged between.73 and.93. Factor analysis and correlations between Nursing Home Physical Performance Test (NHPPT) items and scales with measures of activities of daily living (ADL) suggest that the NHPPT taps aspects of gross motor function (Factor 1) and fine motor coordination and task sequencing (Factor 2) required for ADL function and mobility. The NHPPT may also tap aspects of ADL function and mobility not measured by the MDS (r = -.72-.75), MOSES (r = -.82-.84), or Katz (r = -.75-.77) scales. Effect sizes based on mean change scores were larger for the NHPPT scales (.38-.53) than for the other functional scales (.27-.33). IMPLICATIONS: The NHPPT is a reliable performance-based instrument that discriminates among frail NH residents. Further studies are necessary to assess the value of this instrument for prediction and monitoring of functional status in the NH.

The validation of a mailed health survey for screening of dementia of the Alzheimer's type.

OBJECTIVE: To test the efficacy of a mailed health survey, which included the Clock Completion Test (CCT), to screen previously undiagnosed older adults for dementia of the Alzheimer's type (DAT) in a community-dwelling population, and to determine whether the addition of selected risk factors for Alzheimer's disease (AD) would enhance the screening instrument's operating characteristics. DESIGN: Comparison of the results of a self-administered screen for DAT with the diagnosis of DAT by clinician evaluation or telephone interviews. SETTING: A geriatric assessment clinic. PARTICIPANTS: Three hundred and five women age 65 and older from St. Louis, Missouri. MEASUREMENTS: The sensitivity and specificity of the CCT and the CCT plus risk factors for AD were evaluated using two standards: The Short Blessed Test (SBT) and the physician diagnosis of probable AD using National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria. RESULTS: Sensitivity and specificity for the CCT by SBT criteria were 63% and 79%, respectively. Using the physician's assessment as a criterion, the CCT had better sensitivity (67%) but poorer specificity (69%). Adding two or more risk factors for AD improved sensitivity and specificity to 71% and 89% and, in the physician assessment subgroup, to 75% and 87%, primarily by halving the false-positive rate. CONCLUSION: The combination of the simple, self-administered CCT and two or more AD risk factors is a more effective screening instrument for DAT and potentially preclinical DAT than the CCT alone. However, the instrument may be better suited for use in an office setting because of a poor response rate by subjects with dementia.

Developing multidisciplinary profiles in a day hospital.

The Ferguson Day Hospital provides a patient-centred, multidisciplinary service for patient assessment and rehabilitation. The authors describe the development of a patient profile that has centralised documentation and reflects the multidisciplinary nature of the services provided.

Working memory, metacognitive uncertainty, and belief bias in syllogistic reasoning.

Studies of syllogistic reasoning have shown that the size of the belief bias effect varies with manipulations of logical validity and problem form. This paper presents a mental models-based account, which explains these findings in terms of variations in the working-memory demands of different problem types. We propose that belief bias may reflect the use of a heuristic that is applied when a threshold of uncertainty in one's processing--attributable to working-memory overload--is exceeded during reasoning. Three experiments are reported, which tested predictions deriving from this account. In Experiment 1, conclusions of neutral believability were presented for evaluation, and a predicted dissociation was observed in confidence ratings for responses to valid and invalid arguments, with participants being more confident in the former. In Experiment 2, an attempt to manipulate working-memory loads indirectly by varying syllogistic figure failed to produce predicted effects upon the size of the belief bias effect. It is argued that the employment of a conclusion evaluation methodology minimized the effect of the figural manipulation in this experiment. In Experiment 3, participants' articulatory and spatial recall capacities were calibrated as a direct test of working-memory involvement in belief bias. Predicted differences in the pattern of belief bias observed between high and low spatial recall groups supported the view that limited working memory plays a key role in belief bias.

Bridging the gap: A set of selective 1H-15N-correlations to link sequential neighbors of prolines.

Triple-resonance experiments are standard in the assignment of protein spectra. Conventional assignment strategies use 1H-15N-correlations as a starting point and therefore have problems when proline appears in the amino acid sequence, which lacks a signal in these correlations. Here we present a set of amino acid selective pulse sequences which provide the information to link the amino acid on either side of proline residues and thus complete the sequential assignment. The experiments yield amino acid type selective 1H-15N-correlations which contain signals from the amino protons of the residues either preceding or following proline in the amino acid sequence. These protons are correlated with their own nitrogen or with that of the proline. The new experiments are recorded as two-dimensional experiments and their performance is demonstrated by application to a 115-residue protein domain.

Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity.

The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize 'FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1-peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K(d)s by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer.

The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1beta protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1beta protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1beta proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation.

A simple test of central processing speed: an extension of the Short Blessed Test.

OBJECTIVE: To evaluate the efficacy and reliability of a simple measure of central processing speed: the time to recite the months of the year in reverse order, from the Short Blessed Test of Orientation, Concentration, and Memory (SBT). DESIGN: Cross-sectional and longitudinal designs were used to establish validity and test-retest reliability. SETTING: Participants' homes and by telephone interview. PARTICIPANTS: An age-stratified sample of 120 community-dwelling women, aged 67-94, randomly selected from Medicare recipients of the St. Louis Metropolitan Statistical Area. MEASUREMENTS: Dependent variables were the SBT, the Trail Making Test, computer-generated simple and choice reaction time, and time to say the months of the year backward (TMYB). The independent variable was age. RESULTS: Significant Pearson product-moment correlations were obtained for Trail Making and TMYB with a simple and choice reaction time after controlling for age and cognitive status. An exponential relationship was observed between age and TMYB, expressed both cross-sectionally and as rate of change. Test-retest reliability for TMYB was 0.90. CONCLUSIONS: TMYB is a valid and reliable measure of central processing speed that compares favorably to the more elaborate and time-consuming Trail Making B test. Because of its simplicity and ease of administration, this test provides the clinician with a practical measure of central processing speed. TMYB extends the utility of the widely used Short Blessed Test by measuring this additional and important domain of brain function.

Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: potential for NMR structure determination of large proteins.

NMR investigations of larger macromolecules (> 20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins.

The origin of the alpha-domain intermediate in the folding of hen lysozyme.

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with quenched-flow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to compare the refolding kinetics of hen egg-white lysozyme at 20 degrees C and 50 degrees C. At 50 degrees C there is clear evidence for distinct fast and slow refolding populations, as observed at 20 degrees C, although folding occurs significantly more rapidly. The folding process is, however, substantially more cooperative at the higher temperature. In particular, the transient intermediate on the major refolding pathway at 20 degrees C, having persistent native-like structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labelling at 50 degrees C. In addition, the characteristic maximum in negative ellipticity and the minimum in fluorescence intensity observed in far UV CD and intrinsic fluorescence experiments at 20 degrees C, respectively, are not seen at 50 degrees C. Addition of 2 M NaCl to the refolding buffer at 50 degrees C, however, regenerates both the hydrogen exchange and optical properties associated with the alpha-domain intermediate but has no significant effect on the overall refolding kinetics. Together with previous findings, these results indicate that non-native interactions within the alpha-domain intermediate are directly responsible for the unusual optical properties observed during refolding, and that this intermediate accumulates as a consequence of its intrinsic stability in a folding process where the formation of stable structure in the beta-domain constitutes the rate-limiting step for the majority of molecules.

Structure of the chromatin binding (chromo) domain from mouse modifier protein 1.

The structure of a chromatin binding domain from mouse chromatin modifier protein 1 (MoMOD1) was determined using nuclear magnetic resonance (NMR) spectroscopy. The protein consists of an N-terminal three-stranded anti-parallel beta-sheet which folds against a C-terminal alpha-helix. The structure reveals an unexpected homology to two archaebacterial DNA binding proteins which are also involved in chromatin structure. Structural comparisons suggest that chromo domains, of which more than 40 are now known, act as protein interaction motifs and that the MoMOD1 protein acts as an adaptor mediating interactions between different proteins.

Geropsychiatric nursing knowledge--a need in home care.

Few nursing schools include in their curriculum home care work; fewer still include coursework in geropsychiatric issues. Yet this is a vital area of knowledge, particularly for home care nurses. What might such courses cover, and how important are they?

Zinc co-ordination in the DNA-binding domain of the yeast transcriptional activator PPR1.

The structure of the native zinc form of the DNA binding domain in the yeast transcriptional activator PPR1 was investigated by extended X-ray absorption fine structure (EXAFS). By carrying out the EXAFS measurements at 11k we were able to demonstrate explicitly the proximity of the two zinc ions (Zn-Zn distance = 3.16 +/- 0.03 A) and the presence of bridging cysteine ligands. The results show that the six cysteine residues co-ordinate two zinc ions in a two-metal ion cluster. PPR1 is the first member of this class of protein for which such information has been obtained.

Simulated aged performance on the embedded figures test.

According to Labouvie-Vief (1985) the predominant explanatory mechanism for performance decline among the elderly incorporates a regression-oriented view of cognitive development: The present study tested the hypothesis that age-related performance decline may be explained by the quantitative changes in lower-level visual functions, such as those due to preretinal aging, which preclude the expression of intact cognitive (qualitative) processes. Thirty undergraduate female volunteers (18 to 25 years) viewed the Embedded Figures Test (EFT) under three conditions: no-filter viewing, .6 neutral-density filter viewing, and simulated-aged (yellow-filter plus .5 neutral-density filter) viewing. A one-way ANOVA revealed significant viewing condition effects for the number of items solved, the number of errors, and time-to-solution. Significant linear trends were found for levels of luminous transmittance. Results supported the hypothesis that preretinal aging appears to degrade EFT performance.

The effects of color and preretinal aging on embedded figures test performance: a failure to replicate.

This study examined the hypothesis that preretinal aging may adversely affect performance in older adults. The relationship between preretinal aging and a perceptual variable (i.e., color) was also explored. Under standard testing procedures, the (a) normal, (b) light-attenuated, and (c) simulated aged performance of 90 young women was compared to the performance of 30 elderly women, using two forms (colored or noncolored) of the Embedded Figures Test (EFT). Results revealed that young women performed significantly better than elderly women on all measures, suggesting that neither the simulation of preretinal aging nor color was a significant factor in EFT performance. Inasmuch as the present study failed to replicate Ball and Pollack (1989), the critical age-related factors in cognitive/perceptual performance have yet to be identified.