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The catalytic cycle of beta -lactam synthetase observed by x-ray crystallographic snapshots.

The catalytic cycle of the ATP/Mg(2+)-dependent enzyme beta-lactam synthetase (beta-LS) from Streptomyces clavuligerus has been observed through a series of x-ray crystallographic snapshots. Chemistry is initiated by the ordered binding of ATP/Mg(2+) and N(2)-(carboxyethyl)-l-arginine (CEA) to the apoenzyme. The apo and ATP/Mg(2+) structures described here, along with the previously described CEA.alpha,beta-methyleneadenosine 5'-triphosphate (CEA.AMP-CPP)/Mg(2+) structure, illuminate changes in active site geometry that favor adenylation. In addition, an acyladenylate intermediate has been trapped. The substrate analog N(2)-(carboxymethyl)-l-arginine (CMA) was adenylated by ATP in the crystal and represents a close structural analog of the previously proposed CEA-adenylate intermediate. Finally, the structure of the ternary product complex deoxyguanidinoproclavaminic acid (DGPC).AMP/PP(i)/Mg(2+) has been determined. The CMA-AMP/PP(i)/Mg(2+) and DGPC.AMP/PP(i)/Mg(2+) structures reveal interactions in the active site that facilitate beta-lactam formation. All of the ATP-bound structures differ from the previously described CEA.AMP-CPP/Mg(2+) structure in that two Mg(2+) ions are found in the active sites. These Mg(2+) ions play critical roles in both the adenylation and beta-lactamization reactions.

Adenosine Triphosphate↗

A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway.

It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.

Biotin↗

A 1.2-A snapshot of the final step of bacterial cell wall biosynthesis.

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of beta-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-A resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type D-alanyl-D-alanine carboxypeptidase/transpeptidase (EC ) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

Acylation↗

Structural snapshots of Escherichia coli histidinol phosphate phosphatase along the reaction pathway.

HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of l-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn(2+) ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg(2+), Mn(2+), Co(2+), or Zn(2+), but Ca(2+) has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg(2+) ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca(2+).

Amino Acid Sequence↗

A laboratory evaluation of the accuracy and precision of the photovac snapshot portable gas chromatograph and the Dräger Chip Measurement System monitor for benzene in air measurements.

A laboratory evaluation of the accuracy and precision of two field instruments, the Photovac Snapshot Gas Chromatograph and the Dräger Chip Measurement System (CMS) Monitor, both capable of specific determination of benzene, was carried out. The evaluation was based on the generation of a test atmosphere of known concentration of benzene in a stainless steel calibration chamber and simultaneous sampling of the test atmosphere by each instrument. At the same time, the chamber atmosphere was continuously monitored by one or two data logging photoionization detector (PID) hydrocarbon analysers. A series of six, 10-minute charcoal tube air samples of the test atmosphere was also collected over the 1-hour run and analyzed by gas chromatography. Nine different concentrations ranging from 0.25 ppm to 8 ppm were used. Accuracy was evaluated using National Institute for Occupational Safety and Health (NIOSH) criteria. The Photovac GC consistently met the NIOSH recommended accuracy criteria of +/- 25 percent at or below 1 ppm of benzene, whereas the Drager CMS results generally fell slightly outside this criteria. Compared against less stringent accuracy criteria of +/- 35 percent, all Photovac GC results were acceptable but about 40 percent of Drager CMS results were not. The precision of Photovac GC (CV +/- 10%) is better than Drager CMS (CV = +/- 20% to 40%). Both instruments are, however, good field instruments provided their limitations are taken into account in their use.

Air Pollutants↗

Large T antigen on the simian virus 40 origin of replication: a 3D snapshot prior to DNA replication.

Large T antigen is the replicative helicase of simian virus 40. Its specific binding to the origin of replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three-dimensional structure of the full-length T antigen double hexamer assembled at its origin of replication by cryoelectron microscopy and single-particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer-hexamer junction mainly at two opposite sites. The structure of the T antigen at the origin of replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA replication is proposed.

Algorithms↗

Escherichia coli K-12: a cooperatively developed annotation snapshot--2005.

The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.

Congresses as Topic↗

Estimating occupational health events in workers with asthma or diabetes: a comparison of diary and snapshot methods.

The study was set up to assess the completeness of reporting different types of work-related health events through 6 month and 1 week diaries and telephone questionnaires relating to individual days. Subjects recruited from hospital asthma or diabetic clinics or from surgical day-case units were randomly assigned to either a full participation arm or to one completing only a 6 month diary. Of the 375 study participants, return rates were 68.8% for 6 month diaries, 81.9% for weekly diaries and 86.5% for researcher-administered questionnaires. Significantly higher rates of self-reported poor work performance, symptoms or changes in medication were estimated by snapshot methods (same-day telephone interviews or one-off weekly diaries) than from diaries completed over a 6 month period. Asthmatics and diabetics attending hospital outpatient clinics were no more likely, however, to report work-related health events in the following 6 months than those attending the same hospital for day surgery.

Adolescent↗

Mapping of the radio frequency magnetic field with a MR snapshot FLASH technique.

A fast method to measure the radio frequency magnetic field intensity distribution during magnetic resonance examinations within objects or patients is proposed. Using a snapshot FLASH sequence, several images are obtained with different transmitter amplitudes for the single prepulse prior to the imaging sequence. The signal intensity at each pixel within a selected slice is diminished by this prepulse, unless the pulse angle is 0 degrees or a multiple of 360 degrees. This condition can be used to measure that transmitter amplitude Umax for each pixel which causes an excitation of 360 degrees by the prepulse. The values of Umax can be visualized in an image and are inversely proportional to the radio frequency magnetic field strength at each position within the image. The method can also be used for in-vivo measurements, where it may be difficult to realize prepulses with sufficient high flip angles. In these cases, signal corresponding to a 180 degree prepulse can be used for calculating Umax.

Electromagnetic Fields↗

Time to stop sniffing the air: snapshot survey.

OBJECTIVE: To determine whether the phrase "sniffing the morning air" is useful in positioning patients for tracheal intubation. DESIGN: Snapshot survey. SETTING: Teaching hospital. PARTICIPANTS: 21 anaesthetists. MAIN OUTCOME MEASURES: Angles of neck, line of sight, and hip to shoulder, as measured on a photograph. RESULTS: Angles ranged widely, especially line of sight: 7 degrees -37 degrees with horizontal. CONCLUSIONS: As patients are positioned supine, rather than standing, the phrase is unhelpful; an explicit description of head position would be better.

Anesthesiology↗

Variation of contrast between different brain tissues with an MR snapshot technique.

The lack of tissue contrast in gradient-echo sequences with very short repetition times can be overcome by using a single inversion pulse prior to the entire imaging sequence. The contrast can be changed by variation of the time delay between the inversion pulse and the first excitation. This snapshot technique was introduced for very fast acquisition of images, but it also provides images with a strong contrast of gray to white matter and of brain tissue to cerebrospinal fluid. The authors designed a sequence that allows sufficient spatial resolution and good image quality. The measurement time is 1.2 seconds, and the in-plane resolution is 0.8 mm. A large variation in contrast with different inversion times was demonstrated on brain images of volunteers and patients.

Brain↗

Evaluation of myocardial perfusion abnormalities with gadolinium-enhanced snapshot MR imaging in humans. Work in progress.

To determine whether myocardial perfusion abnormalities could be detected in patients with coronary artery disease by means of contrast material-enhanced magnetic resonance (MR) images, a snapshot imaging technique was used in six patients with coronary artery disease and four healthy subjects in conjunction with pharmacologic stress (dipyridamole infusion) and bolus injection of gadopentetate dimeglumine. MR images from all patients and healthy subjects were quantitatively analyzed to define spatial changes in signal intensity after administration of dipyridamole and gadopentetate dimeglumine. The resultant findings were compared with findings on thallium-201 scintigrams obtained after administration of dipyridamole and on coronary arteriograms in all patients. Nine myocardial regions supplied by stenosed arteries showed diminished levels of signal intensity after infusion of the contrast agent compared with those of normally perfused regions. These findings were in agreement with those obtained with T1-201 scintigraphy (in eight of nine regions) and arteriography. Thus, contrast-enhanced high-speed MR imaging with use of dipyridamole enabled detection of regional perfusion abnormalities in humans.

Adult↗

Transcriptomic analysis of the cardiac left ventricle in a rodent model of diabetic cardiomyopathy: molecular snapshot of a severe myocardial disease.

Heart disease is the major cause of death in diabetes, a disorder characterized by chronic hyperglycemia and cardiovascular complications. Diabetic cardiomyopathy (DCM) is increasingly recognized as a major contributor to diastolic dysfunction and heart failure in diabetes, but its molecular basis has remained obscure, in part because of its multifactorial origins. Here we employed comparative transcriptomic methods with quantitative verification of selected transcripts by reverse transcriptase quantitative PCR to characterize the molecular basis of DCM in rats with streptozotocin-induced diabetes of 16-wk duration. Diabetes caused left ventricular disease that was accompanied by significant changes in the expression of 1,614 genes, 749 of which had functions assignable by Gene Ontology classification. Genes corresponding to proteins expressed in mitochondria accounted for a disproportionate number of those whose expression was significantly modified in DCM, consistent with the idea that the mitochondrion is a key target of the pathogenic processes that cause myocardial disease in diabetes. Diabetes also induced global perturbations in the expression of genes regulating cardiac fatty acid metabolism, whose dysfunction is likely to play a key role in the promotion of oxidative stress, thereby contributing to the pathogenesis of diabetic myocardial disease. In particular, these data point to impaired regulation of mitochondrial beta-oxidation as central in the mechanisms that generate DCM pathogenesis. This study provides a comprehensive molecular snapshot of the processes leading to myocardial disease in diabetes.

Animals↗

The special/general education integration initiative for students with specific learning disabilities: a "snapshot" of program change.

As part of a broader investigation, this study sought to (a) provide a statewide "snapshot" of educators' views regarding the current status of and process associated with increased integration efforts for serving students with specific learning disabilities (SLD) in Virginia, and (b) document factors serving as the basis of or creating reluctance toward such efforts. Survey respondents included the population of special education supervisors and samples of general education supervisors, building principals, general secondary and elementary education teachers, and LD teachers (N = 788). Results suggest active efforts to increase the amount of time students with SLD spend in general classroom settings; however, limited program change-related guidelines or category-specific outcome-monitoring measures were reported. Across groups, participants expressed doubts regarding the adequacy of general education teachers' skills for making needed instructional adaptations. Over half of the respondents tended to disagree or disagreed that general education teachers were willing to make needed adaptations for students with SLD. Differences in the views expressed by the different respondent groups were examined.

Data Collection↗

A snapshot view of the impact of chronic pain on adolescents.

This study provides a snapshot view on the impact that chronic pain has on the daily functioning of a small group of adolescents (n = 10). Chronic, severe pain in childhood and adolescence is not common, but is increasingly recognized to occur and be worthy of treatment. Referrals come from a variety of sources including paediatric orthopaedic surgeons, paediatric rheumatologists, paediatric neurologists, child psychiatrists, and GPs. Examples of the types of conditions seen are complex regional pain syndromes, low back pain, pain caused by congenital or degenerative disease and chronic headaches. Additionally, chronic pain affects the well-being of the family as well as the adolescent. Many adolescents have been unable to attend school for months or years, and their educational and social development is impaired. The aim of treatment is to rehabilitate the adolescents within the context of their families and to return them to school and to participating in normal social activities within the limits of their condition.

Adolescent↗

Femtosecond snapshot imaging of propagating light itself.

An ultrafast imaging technique has been developed to visualize directly a light pulse that is propagating in a medium. The method, called femtosecond time-resolved optical polarigraphy (FTOP), senses instantaneous changes in the birefringence within the medium that are induced by the propagation of an intense light. A snapshot sequence composed of each femtosecond probing the pulse delay enables ultrafast propagation dynamics of the intense femtosecond laser pulse in the medium, such as gases and liquids, to be visualized directly. Other examples include the filamentation dynamics in CS2 liquid and the propagation dynamics in air related to the interaction with laser breakdown plasma. FTOP can also be used to extract information on the optical Kerr constant and its decay time in media. This method is useful in the monitoring of the intensity distribution in the nonlinear propagation of intense light pulses, which is a frequently studied subject in the field of physics regarding nonlinear optics and laser processing.

Journal Article↗

Spatial-spectral modulating snapshot hyperspectral imager.

Experimental results are presented for a computed tomography imaging spectrometer (CTIS) with imposed spatial-spectral modulation on the image scene. This modulation structure on the CTIS tomographic dispersion created substantial gains in spectral reconstruction resolution after standard iterative, nonlinear, inversion techniques were used. Modulation limits system ambiguities, so high-frequency spectral and low-frequency spatial scene data could be recovered. The results demonstrate how spatial modulation acts as a high-frequency spectral deconvolver for the snapshot hyperspectral imager technology.

Journal Article↗

Modeling and optimization for a prismatic snapshot imaging polarimeter.

Thin birefringent prisms placed near an image plane introduce sinusoidal fringes onto a 2D polarized scene making possible a snapshot imaging polarimeter, which encodes polarization information into the modulation of the fringes. This approach was introduced by Oka and Kaneko [Opt. Express 11, 1510 (2003)], who analyzed the instrument through the Mueller calculus. We show that the plane-wave assumption adopted in the Mueller theory can introduce unnecessary error in a polarimeter design. To directly take prism effects such as beam splitting and deviating into accounts we introduce a geometric imaging model, which allows for a versatile simulation of the birefringent prisms and provides a means for optimization. A calcite visible system is investigated as an example, which essentially shows how each design parameter affects the overall image quality and how to modify the polarimeter design to optimize overall performance. The approach is applicable to any prismatic imaging polarimeter with different prism materials and different working wavelengths.

Journal Article↗