Multiple protein structure alignment

A method was developed to compare protein structures and to combine them into a multiple structure consensus. Previous methods of multiple structure comparison have only concatenated pairwise alignments or produced a consensus structure by averaging coordinate sets. The current method is a fusion of the fast structure comparison program SSAP and the multiple sequence alignment program MULTAL. As in MULTAL, structures are progressively combined, producing intermediate consensus structures that are compared directly to each other and all remaining single structures. This leads to a hierarchic "condensation," continually evaluated in the light of the emerging conserved core regions. Following the SSAP approach, all interatomic vectors were retained with well-conserved regions distinguished by coherent vector bundles (the structural equivalent of a conserved sequence position). Each bundle of vectors is summarized by a resultant, whereas vector coherence is captured in an error term, which is the only distinction between conserved and variable positions. Resultant vectors are used directly in the comparison, which is weighted by their error values, giving greater importance to the matching of conserved positions. The resultant vectors and their errors can also be used directly in molecular modeling. Applications of the method were assessed by the quality of the resulting sequence alignments, phylogenetic tree construction, and databank scanning with the consensus. Visual assessment of the structural superpositions and consensus structure for various well-characterized families confirmed that the consensus had identified a reasonable core.     

Reduced mechanical efficiency in chronic obstructive pulmonary disease but normal peak VO2 with small muscle mass exercise

We studied six patients with chronic obstructive pulmonary disease (COPD) (FEV1 = 1.1 +/- 0.2 L, 32% of predicted) and six age- and activity level-matched control subjects while performing both maximal bicycle exercise and single leg knee-extensor exercise. Arterial and femoral venous blood sampling, thermodilution blood flow measurements, and needle biopsies allowed the assessment of muscle oxygen supply, utilization, and structure. Maximal work rates and single leg VO2max (control subjects = 0.63 +/- 0.1; patients with COPD = 0.37 +/- 0.1 L/minute) were significantly greater in the control group during bicycle exercise. During knee-extensor exercise this difference in VO2max disappeared, whereas maximal work capacity was reduced (flywheel resistance: control subjects = 923 +/- 198; patients with COPD = 612 +/- 81 g) revealing a significantly reduced mechanical efficiency (work per unit oxygen consumed) with COPD. The patients had an elevated number of less efficient type II muscle fibers, whereas muscle fiber cross-sectional areas, capillarity, and mitochondrial volume density were not different between the groups. Therefore, although metabolic capacity per se is unchanged, fiber type differences associated with COPD may account for the reduced muscular mechanical efficiency that becomes clearly apparent during knee-extensor exercise, when muscle function is no longer overshadowed by the decrement in lung function.     

Stages of Change or Changes of Stage? Predicting Transitions in Transtheoretical Model Stages in Relation to Healthy Food Choice.

Relatively little research has examined factors that account for transitions between transtheoretical model (TTM) stages of change. The present study (N=787) used sociodemographic, TTM, and theory of planned behavior (TPB) variables, as well as theory-driven interventions to predict changes in stage. Longitudinal analyses revealed that sociodemographic, TPB, and 1 of the interventions predicted transitions between most stages of change. In fact, only progression from the preparation stage was not predictable. However, given that this change of stage marks the transition between cognition and actual behavior, the identification of variables that bridge this gap is crucial for the development of interventions to promote stage transitions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Glucoamylase mutants in the conserved active-site segment Trp170-Tyr175 located at a distance from the site of catalysis

To mimic the structure of the 1.8-fold more active (k(cat)) Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA was subjected to site- directed mutagenesis in the Trp170-Tyr175 segment of the third of the six well-conserved alpha-->alpha connecting loops of the catalytic (alpha/alpha)6-barrel. While the Trp170-->Phe, Gln172-->Asn and Tyr175-- >Phe mutants showed an up to 1.7-fold increased k(cat) and Gly174-->Cys GA and approximately 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171-->Ser, Thr173-->Gly and A.niger wild-type GA had very similar kcat and K(m) values for the hydrolysis of isomaltose and the malto-oligosaccharides of DP 2-7. Crystal structures of pseudotetrasaccharide inhibitor complexes of Aspergillus awamori var. X100 GA, which is 94% identical to A.niger GA, indicate that Tyr175 is located at binding subsite 4, while the preceding target residues and the high-mannose type unit on Asn171 are at a larger distance from the site of catalysis. The mutations had a modest effect on thermostability; the temperature for 50% inactivation, Tm, was thus unchanged for Tyr175 -->Phe GA and reduced by 0.2-2.9 degrees C for the other mutants. The deletion of the N-linked high-mannose unit-in Asn171 -->Ser and Thr173-->Gly GAs-appeared to be of minor importance for enzyme activity and thermostability, and did not increase the sensitivity to proteolysis.      

The infected urachal cyst: primary excision versus a staged approach

PURPOSE: We compared outcomes following single or 2-stage repair of infected urachal cysts in the pediatric population. MATERIALS AND METHODS: We reviewed the records of 17 patients 1 day to 14 years old (median age 22 months) with a urachal cyst. Immediate cyst excision was performed in 6 patients without infection, while those with an abscess underwent single or 2-stage repair. RESULTS: Median postoperative hospital stay for the urachal abscess group was 14 and 11.5 days for single and 2-stage procedures, respectively. After immediate excision postoperative complications developed in each case, although none occurred with a 2-stage approach. CONCLUSIONS: In the absence of infection, urachal cyst excision affords the most benign postoperative course. However, when infection is present, perioperative drainage with subsequent total excision, including a cuff of bladder, may offer the most effective surgical option.     

Advanced multiple beam equalization radiography (AMBER) combined with computed radiography. Preliminary evaluation

The combined use of AMBER (Advanced Multiple Beam Equalization Radiography) and a digital storage phosphor (SP) radiography system was evaluated for chest radiography in a pilot study with 4 patients. Four image modes with different dose levels were compared: the SP in combination with an AMBER equalized exposure (SP/AMBER) and 3 nonequalized exposures with dose levels corresponding to the respective calculated AMBER lung dose (SP/lung field dose), the calculated AMBER mediastinal dose (SP/mediastinal dose) and the calculated AMBER average dose (SP/average dose). All image modes were matched for Hurter and Driffield characteristics and subjectively rated according to visibility of details. The improved signal-to-noise (S/N) ratio of SP/AMBER resulted in a better visualization of structures in the mediastinum and the basal lung where SP/lung field dose scored lowest. For the central lung no quality differences were seen between techniques. The compressed dynamic range of the SP/AMBER images was more easily displayed on the hard-copy film. The combination of AMBER with SP radiography promises to overcome the dynamic range limitations of digital displays while, at moderate doses, giving better S/N and image quality than standard SP technique.     

Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits

To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LC1 and LC2, and two size forms of the class D L-type alpha 1 subunit, LD1 and LD2. The larger isoforms had apparent molecular masses of approximately 200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5% acrylamide. Immunocytochemical studies using CNC1 and CND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.     

High performance thin-layer chromatographic method for the determination of sparfloxacin in human plasma and its use in pharmacokinetic studies

A rapid and sensitive high-performance thin-layer chromatographic (HPTLC) method has been developed for the measurement of sparfloxacin in human plasma and its use for pharmacokinetic study has been evaluated. Detection and quantitation were performed without using an internal standard. A single stage extraction procedure was followed for extracting sparfloxacin from plasma and a known amount of the extract was spotted on precoated silica gel 60 F254 plates using a Camag Linomat IV autosampler. Sparfloxacin was quantified using a Camag TLC Scanner 3. The recovery study of authentic analytes added to plasma at 0.1 to 0.8 microgram ml-1 was 94.9 +/- 0.98% and the lowest amount of sparfloxacin that could be detected was 50 ng ml-1 plasma. The method provides a direct estimate of the amount of sparfloxacin present in plasma. The method was used for the determination of plasma levels as well as pharmacokinetic parameters of sparfloxacin after oral administration of two marketed preparations to healthy volunteers.