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At least 145 records · Page 8Linked to original sources
PubMed2000

Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding I: analysis of cro dimerization from nanomolar to micromolar concentrations.

The cro repressor from bacteriophage lambda is an important and classical transcription regulatory protein that binds DNA operator sites as a dimer. Therefore, a complete understanding of gene regulation by cro requires knowledge of the coupled energetics of its protein dimerization and site-specific DNA binding. A method is described by which cro repressor can be labeled in vivo with [(35)S]methionine to a specific activity of 2 x 10(15) cpm/mol. As a prelude to binding studies, the association equilibrium of cro was determined over the range 10(-)(9)-10(-)(3) M using large-zone analytical gel chromatography with radiolabeled repressor. The data are best described by a monomer-dimer stoichiometry with an equilibrium constant of 3.07 (+/-1.08) x 10(6) M(-)(1) total cro monomer. Stokes radii for monomers and dimers were evaluated from the resolved gel partition coefficients. Under the conditions employed in this study (10 mM Bis-Tris, 200 mM KCl, 2.5 mM MgCl(2), 1 mM CaCl(2), 100 microg/mL BSA, pH 7.0, 20 degrees C), self-association of cro to species with assembly states greater than dimers is not observed.

Bacteriophage lambda↗
PubMed2006

Pseudolinear gradient ultrahigh-pressure liquid chromatography using an injection valve assembly.

The use of ultrahigh pressures in liquid chromatography (UHPLC) imposes stringent requirements on hardware such as pumps, valves, injectors, connecting tubing, and columns. One of the most difficult components of the UHPLC system to develop has been the sample injector. Static-split injection, which can be performed at pressures up to 6900 bar (100,000 psi), consumes a large sample volume and is very irreproducible. A pressure-balanced injection valve provided better reproducibility, shorter injection time, reduced sample consumption, and greater ease of use; however, it could only withstand pressures up to approximately 1000 bar (15,000 psi). In this study, a new injection valve assembly that can operate at pressures as high as 2070 bar (30,000 psi) was evaluated for UHPLC. This assembly contains six miniature electronically controlled needle valves to provide accurate and precise volumes for introduction into the capillary LC column. It was found that sample volumes as small as several tenths of a nanoliter can be injected, which are comparable to the results obtained from the static-split injector. The reproducibilities of retention time, efficiency, and peak area were investigated, and the results showed that the relative standard deviations of these parameters were small enough for quantitative analyses. Separation experiments using the UHPLC system with this new injection valve assembly showed that this new injector is suitable for both isocratic and gradient operation modes. A newly designed capillary connector was used at a pressure as high as 2070 bar (30,000 psi).

Journal Article↗
PubMed1998

Crystal structures of MS2 coat protein mutants in complex with wild-type RNA operator fragments.

In MS2 assembly of phage particles results from an interaction between a coat protein dimer and a stem-loop of the RNA genome (the operator hairpin). Amino acid residues Thr45, which is universally conserved among the small RNA phages, and Thr59 are part of the specific RNA binding pocket and interact directly with the RNA; the former through a hydrogen bond, the latter through hydrophobic contacts. The crystal structures of MS2 protein capsids formed by mutants Thr45Ala and Thr59Ser, both with and without the 19 nt wild-type operator hairpin bound, are reported here. The RNA hairpin binds to these mutants in a similar way to its binding to wild-type protein. In a companion paper both mutants are shown to be deficient in RNA binding in an in vivo assay, but in vitro the equilibrium dissociation constant is significantly higher than wild-type for the Thr45Ala mutant. The change in binding affinity of the Thr45Ala mutant is probably a direct consequence of removal of direct hydrogen bonds between the protein and the RNA. The properties of the Thr59Ser mutant are more difficult to explain, but are consistent with a loss of non-polar contact.

Base Sequence↗
PubMed1984

The use of touch-sensitive screen in interactive morphometry.

We propose the use of a touch-sensitive screen as a tool to facilitate acquisition of data both in point counting and morphometric planimetry. It obviates the use of a 'correspondence image' required with many planimetric devices in which the means for data acquisition and display are separate. The touch-sensitive screen offers operational advantages over the light pen. The characteristics of this new graphical input device, its assembly and basic operational considerations are introduced and discussed.

Computers↗
PubMed1996

Functional measuring of muscle tone.

Testing muscle fields requires functional conditions. The procedure for measuring has to put the musculature under conditions as they occur in physiological functioning. Furthermore a measuring instrument should be economical and easy to handle in the course of the daily routine. The Myofunctional Therapist presently has a number of measuring instruments available. Some of them need to be adjusted before every operation. Others give only comparative readings, having no absolute reference. Others measure states which do not represent normal functions of the musculature and therefore do not allow conclusions about the state of the muscle in relation to function. Some apparatuses are highly sensitive and can only be handled by highly trained persons. In this category we find the highly precise electronic equipment being highly expensive to purchase and to repair. It seems that the Function Tonometer is in all points compatible when waging the pros and cons. It directly reads the actual muscle tone under conditions which occur during normal function. The device can be used for diagnostics and documentation as well as in functional training. The output is a defined mechanical range (pressure in mbar) and needs not be adjusted before each measurement. Used in treatment, it motivates by visual feedback. The results are reproducible at any time, at any location, and by any person, even young patients. The instrument has an easy handling, favorable costs in purchase and operation, and is not easily damaged. The assembly is not electrically operated and thus is not under restrictive rules nor the need of an energy supply. The Function Tonometer is indeed a flexible and useful tool to aid in diagnosis, documentation, and treatment procedures.

Equipment Design↗
PubMed2001

Enhanceosomes.

Gene-specific transcriptional regulation in higher eukaryotes is mediated by complex cis-acting control elements that specify the location, timing and magnitude of the response. During the past five years, an argument has been made that in several cases specificity in gene transcription is achieved by the assembly of higher-order three-dimensional transcription factor/enhancer DNA complexes, termed enhanceosomes. The inherent co-operativity in enhanceosome assembly and the embedded synergy in transcription ensure that a specific gene would be selected for activation only if all the enhanceosome components are present in the same nucleus. Enhanceosomes activate transcription by recruiting chromatin-modifying activities and basal transcription factors to the nearby promoters.

Animals↗
PubMed1993

Basement membrane and interstitial matrix components form separate matrices in heterokaryons of PYS-2 cells and fibroblasts.

In order to gain further understanding of the spatial organization of interstitial and basement membrane matrices, we studied the expression of the interstitial matrix protein, fibronectin, and the basement membrane protein, laminin, in heterokaryons formed by the fusion of normal fibroblasts and teratocarcinoma-derived epithelial PYS-2 cells. These heterokaryons showed various distributions of the matrix proteins depending on the proportions of the different parental cell nuclei within the cytoplasm of the cell. Heterokaryons containing equal numbers of fibroblast and PYS-2 cell nuclei showed an abundant laminin matrix subcellularly and only minor amounts of fibronectin matrix at the periphery of the cells. Similar results were obtained in heterokaryons containing an excess of epithelial cell nuclei. In heterokaryons containing an excess of fibroblast nuclei, on the other hand, laminin matrix was reduced and a fibrillar fibronectin matrix was seen also on top of the cell body. The results suggest a gene dosage-type of effect on the expression of these proteins. Furthermore, extracellular laminin and fibronectin matrices did not codistribute around the heterokaryons but the two proteins were assembled into separate structures. The lack of codistribution of fibronectin and laminin matrices in heterokaryons suggests that the molecular interactions, which determine the assembly of basement membrane and interstitial matrices in these cells are highly type-specific. Similar mechanisms may also operate in the assembly of extracellular matrices in vivo.

3T3 Cells↗
PubMed2006

Complications associated with an implantable vascular access device.

PURPOSE: Implantable vascular access devices (ports) are well accepted in the management of many pediatric conditions. Modifications have improved port function, patient satisfaction, and enhanced compatibility with imaging studies. We reviewed our experience with a port system and identified unique mechanical complications. METHODS: From 1998 to the present, 301 patients underwent 296 port insertions and 175 port removals. We assessed medical records, radiographs, and operative findings. The 6.6F MRI Low-Profile Implanted Port (Bard Access Systems, Salt Lake City, Utah) was used almost exclusively and was assembled by the operating surgeon. Outcome measures included port reservoir leakage, catheter dislodgment, and number of device days until complication. Ports were implanted for multiple medical problems including 74.2% in hematology/oncology patients. RESULTS: For 296 port insertions, 15 complications (5.1%) were identified in 13 patients (mean age, 8.4 years). Eleven leaks (3.7%) in 9 patients were found, with 9 leaks resulting from needle perforation of the port base and 2 leaks seen at the catheter connection site. Average port duration was 425 days (range, 12-1266 days) before leakage. Four patients had catheter dislodgment (1.4%), with 3 of 4 catheters embolizing to the heart or pulmonary artery. Patients were asymptomatic, and catheters were retrieved by interventional radiology. Dislodgment at the catheter-port connection site was seen in 3 of 4 cases, and average port duration was 1075 days (range, 269-2657 days) until catheter separation. Twelve of 13 patients had successful implantation of a new port system. CONCLUSIONS: This study identifies that (1) mechanical port complications (5.1%) are not rare for this device; (2) regardless of port age, the thin plastic base may result in a risk of perforation not seen in other devices; (3) the extended period before embolization likely indicates device wear rather than faulty assembly; and (4) complications could be successfully managed including retrieval of embolized catheters.

Adolescent↗
PubMed2003

Surgical robotic applications in otolaryngology.

OBJECTIVES: To explore the feasibility of performing endo-robotic neck surgery in a porcine model and to compare the results of robotically enhanced endoscopic surgery with those from a conventional endoscopic technique. STUDY DESIGN: Prospective, nonrandomized experimental investigation in a porcine model. METHODS: We performed a consecutive series of endoscopic neck surgeries using the daVinci surgical system (Intuitive Surgical Inc.). The length of time required to establish the operative pocket and to assemble the robotic components, as well as the total duration of each operation, was recorded. The animals were continuously monitored for heart rate, blood pressure, and end-tidal CO(2) pressure, and evaluation for presence of pneumothorax and subcutaneous emphysema was undertaken postoperatively. The specimens were examined histologically. RESULTS: Four different types of neck surgery were successfully performed on both sides of the neck of four animals using the daVinci surgical system. Creation of the operative pocket took, on average (+/-SD), 18.1 +/- 11.9 minutes, and assembly of the robot required 12.5 +/- 9.9 minutes, resulting in a mean preparation time for all procedures of 30.6 +/- 21.0 minutes. The mean operative time for submandibular resection (n = 3) was 19.0 +/- 6.6 minutes, with a total procedure time of 39.0 +/- 10.2 minutes. Selective neck dissections (n = 3) required a mean operative time of 66.0 +/- 18.5 minutes and a total procedure time of 85.7 +/- 16.7 minutes. One partial parotidectomy and one thymectomy were also performed. The median estimated blood loss was 0 mL (range, 0-10 mL). The end-tidal CO(2) pressure fell from the start to the end of the procedures by a mean of 4.4 +/- 7.9 mm Hg. The blood pressure fell by a mean of 1.9 +/- 7.5 mm Hg. There was one case of modest subcutaneous emphysema, and there were no cases of pneumothorax or air embolism. No conversions to open resection were necessary. CONCLUSIONS: Robotically enhanced endoscopic surgery in the neck is feasible and offers a number of compelling advantages over conventional endoscopic neck surgery. Clinical trials will be necessary to determine whether these advantages can be achieved in clinical practice.

Animals↗
PubMed2004

The Saccharomyces cerevisiae TRT2 tRNAThr gene upstream of STE6 is a barrier to repression in MATalpha cells and exerts a potential tRNA position effect in MATa cells.

A growing body of evidence suggests that genes transcribed by RNA polymerase III exhibit multiple functions within a chromosome. While the predominant function of these genes is the synthesis of RNA molecules, certain RNA polymerase III genes also function as genomic landmarks. Transfer RNA genes are known to exhibit extra-transcriptional activities such as directing Ty element integration, pausing of replication forks, overriding nucleosome positioning sequences, repressing neighboring genes (tRNA position effect), and acting as a barrier to the spread of repressive chromatin. This study was designed to identify other tRNA loci that may act as barriers to chromatin-mediated repression, and focused on TRT2, a tRNA(Thr) adjacent to the STE6 alpha2 operator. We show that TRT2 acts as a barrier to repression, protecting the upstream CBT1 gene from the influence of the STE6 alpha2 operator in MATalpha cells. Interestingly, deletion of TRT2 results in an increase in CBT1 mRNA levels in MATa cells, indicating a potential tRNA position effect. The transcription of TRT2 itself is unaffected by the presence of the alpha2 operator, suggesting a hierarchy that favors assembly of the RNA polymerase III complex versus assembly of adjacent alpha2 operator-mediated repressed chromatin structures. This proposed hierarchy could explain how tRNA genes function as barriers to the propagation of repressive chromatin.

ATP-Binding Cassette Transporters↗
PubMed2002

A functional interaction between the putative primosomal protein DnaI and the main replicative DNA helicase DnaB in Bacillus.

In Gram negative Escherichia coli there are two well-characterised primosomal assembly processes, the PriA- and DnaA-mediated cascades. The presence of PriA and DnaA proteins in Gram positive Bacillus spp. supports the assumption that both the PriA- and DnaA-mediated primosomal assembly cascades also operate in these organisms. However, the lack of sequence homology between the rest of the primosomal proteins indicates significant differences between these two bacterial species. Central to the process of primosomal assembly is the loading of the main hexameric replicative helicase (DnaB in E.coli and DnaC in Bacillus subtilis) on the DNA. This loading is achieved by specialised proteins known as 'helicase loaders'. In E.coli DnaT and DnaC are responsible for loading DnaB onto the DNA during primosome assembly, in the PriA- and DnaA-mediated cascades, respectively. In Bacillus the identity of the helicase loader is still not established unequivocally. In this paper we provide evidence for a functional interaction between the primosomal protein DnaI from B.subtilis and the main hexameric replicative helicase DnaB from Bacillus stearothermophilus. Our results are consistent with the putative role of DnaI as the 'helicase loader' in the Gram positive Bacillus spp.

Adenosine Triphosphatases↗
PubMed1988

Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA.

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.

Aldehydes↗
PubMed2001

Dynamics of peroxisome assembly and function.

Recent studies in human cells and in the yeast Yarrowia lipolytica have shown that peroxisomes consist of numerous structurally distinct subcompartments that differ in their import competency for various proteins and are related through a time-ordered conversion of one subcompartment to another. Our studies have implicated the fusion of small peroxisomal precursors as an early event in the multistep assembly of peroxisomes operating in Y. lipolytica. Newly discovered unexpected roles for peroxisomes in specific developmental programs have expanded the remarkable plasticity of peroxisomal functions. Here, we highlight recent discoveries on the highly dynamic nature of peroxisome assembly and function and suggest questions for future research in these areas.

Animals↗
PubMed1996

Possible health risks from low level exposure to beryllium.

The first case of chronic beryllium disease (CBD) at the Rocky Flats Environmental Technology Site (Rocky Flats) was diagnosed in a machinist in 1984. Rocky Flats, located 16 miles northwest of Denver, Colorado, is part of the United States Department of Energy (DOE) nuclear weapons complex. Research and development operations using beryllium began at Rocky Flats in 1953, and beryllium production operations began in 1957. Exposures could have occurred during foundry operations, casting, shearing, rolling, cutting, welding, machining, sanding, polishing, assembly, and chemical analysis operations. The Beryllium Health Surveillance Program (BHSP) was established in June 1991 at Rocky Flats to provide health surveillance for beryllium exposed employees using the Lymphocyte Proliferation Test (LPT) to identify sensitized individuals. Of the 29 cases of CBD and 76 cases of beryllium sensitization identified since 1991, several cases appear to have had only minimal opportunistic exposures to beryllium, since they were employed in administrative functions rather than primary beryllium operations. In conjunction with other health surveillance programs, a questionnaire and interview are administered to obtain detailed work and health histories. These histories, along with other data, are utilized to estimate the extent of an individual's exposure. Additional surveillance is in progress to attempt to characterize the possible risks from intermittent or brief exposures to beryllium in the workplace.

Berylliosis↗
PubMed2006

Insights into the distribution of water in a self-humidifying H2/O2 proton-exchange membrane fuel cell using 1H NMR microscopy.

Proton ((1)H) NMR microscopy is used to investigate in-situ the distribution of water throughout a self-humidifying proton-exchange membrane fuel cell, PEMFC, operating at ambient temperature and pressure on dry H(2)(g) and O(2)(g). The results provide the first experimental images of the in-plane distribution of water within the PEM of a membrane electrode assembly in an operating fuel cell. The effect of gas flow configuration on the distribution of water in the PEM and cathode flow field is investigated, revealing that the counter-flow configurations yield a more uniform distribution of water throughout the PEM. The maximum power output from the PEMFC, while operating under conditions of constant external load, occurs when H(2)O(l) is first visible in the (1)H NMR image of the cathode flow field, and subsequently declines as this H(2)O(l) continues to accumulate. The (1)H NMR microscopy experiments are in qualitative agreement with predictions from several theoretical modeling studies (e.g., Pasaogullari, U.; Wang, C. Y. J. Electrochem. Soc. 2005, 152, A380-A390), suggesting that combined theoretical and experimental approaches will constitute a powerful tool for PEMFC design, diagnosis, and optimization.

Journal Article↗
PubMed2006

Concatenated logic gates using four coupled biocatalysts operating in series.

The assembly of three concatenated enzyme-based logic gates consisting of OR, AND, XOR is described. Four biocatalysts, acetylcholine esterase, choline oxidase, microperoxidase-11, and the NAD+-dependent glucose dehydrogenase, are used to assemble the gates. Four inputs that include acetylcholine, butyrylcholine, O2, and glucose are used to drive the concatenated-gates system. The cofactor NAD+, and its reduced 1,4-dihydro form, NADH, are used as a reporter couple, and these provide an optical output for the gates. The modulus of the absorbance changes of NADH is used as a readout signal.

Acetylcholinesterase↗
PubMed2006

Pt and Ru X-ray absorption spectroscopy of PtRu anode catalysts in operating direct methanol fuel cells.

In situ X-ray absorption spectroscopy, ex situ X-ray fluorescence, and X-ray powder diffraction enabled detailed core analysis of phase segregated nanostructured PtRu anode catalysts in an operating direct methanol fuel cell (DMFC). No change in the core structures of the phase segregated catalyst was observed as the potential traversed the current onset potential of the DMFC. The methodology was exemplified using a Johnson Matthey unsupported PtRu (1:1) anode catalyst incorporated into a DMFC membrane electrode assembly. During DMFC operation the catalyst is essentially metallic with half of the Ru incorporated into a face-centered cubic (FCC) Pt alloy lattice and the remaining half in an amorphous phase. The extended X-ray absorption fine structure (EXAFS) analysis suggests that the FCC lattice is not fully disordered. The EXAFS indicates that the Ru-O bond lengths were significantly shorter than those reported for Ru-O of ruthenium oxides, suggesting that the phases in which the Ru resides in the catalysts are not similar to oxides.

Catalysis↗