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This paper describes an effort to bridge research and practice in residential care through implementing a program model titled Children and Residential Experiences (CARE). The strategy involves consulting at all levels of the organization to guide personnel to incorporate CARE evidence-based principles into daily practice, and fostering an organizational culture and climate that sustains the integration of CARE principles. CARE aims to promote residential care programs that serve the best interests of children. [ABSTRACT FROM AUTHOR]

The current study examined the impact of a setting-level intervention on the prevention of aggressive or dangerous behavioral incidents involving youth living in group care environments. Eleven group care agencies implemented Children and Residential Experiences (CARE), a principle-based program that helps agencies use a set of evidence-informed principles to guide programming and enrich the relational dynamics throughout the agency. All agencies served mostly youth referred from child welfare. The 3-year implementation of CARE involved intensive agency-wide training and on-site consultation to agency leaders and managers around supporting and facilitating day-to-day application of the principles in both childcare and staff management arenas. Agencies provided data over 48 months on the monthly frequency of behavioral incidents most related to program objectives. Using multiple baseline interrupted time series analysis to assess program effects, we tested whether trends during the program implementation period declined significantly compared to the 12 months before implementation. Results showed significant program effects on incidents involving youth aggression toward adult staff, property destruction, and running away. Effects on aggression toward peers and self-harm were also found but were less consistent. Staff ratings of positive organizational social context (OSC) predicted fewer incidents, but there was no clear relationship between OSC and observed program effects. Findings support the potential efficacy of the CARE model and illustrate that intervening "upstream" at the setting level may help to prevent coercive caregiving patterns and increase opportunities for healthy social interactions.

Human cytomegalovirus (CMV), classified as human herpesvirus 5, is ubiquitous in human populations. Infection generally causes little illness in healthy individuals, but can cause life-threatening disease in those who are immunocompromised or in newborns through complications arising from congenital CMV infection. An important aspect in diagnosis and treatment is to track circulating viral load with molecular methods, particularly with quantitative PCR. Standardization is vital, because of interlaboratory variability (due in part to the variety of assays and calibrants). Toward that end, the U.S. National Institute of Standards and Technology produced a Standard Reference Material 2366 appropriate for establishing metrological traceability of assay calibrants. This standard is composed of CMV DNA (Towne(Δ147) bacterial artificial chromosome DNA). Regions of the CMV DNA that are commonly used as targets for PCR assays were sequenced. Digital PCR was used to quantify the DNA, with concentration expressed as copies per microliter. The materials were tested for homogeneity and stability. An interlaboratory study was conducted by Quality Control for Molecular Diagnostics (Glasgow, UK), in which one component of SRM 2366 was included for analysis by participants in a CMV external quality assessment and proficiency testing program., (Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

There is a great need for harmonization in nucleic acid testing for infectious disease and clinical genetics. The proliferation of assay methods, the number of targets for molecular diagnostics and the absence of standard reference materials contribute to variability in test results among laboratories. This article provides a comprehensive overview of reference materials, related documentary standards and proficiency testing programs. The article explores the relationships among these resources and provides necessary information for people practicing in this area that is not taught in formal courses and frequently is obtained on an ad hoc basis. The aim of this article is to provide helpful tools for molecular diagnostic laboratories.

An online survey was conducted by the International Life Sciences Institute, Food Biotechnology Committee, on the use of qualitative and quantitative polymerase chain reaction (PCR) assays for cauliflower mosaic virus 35S promoter and Agrobacterium tumefaciens Tnos DNA sequence elements for the detection of genetically engineered (GE) crop plant material. Forty-four testing laboratories around the world completed the survey. The results showed the widespread use of such methods, the multiplicity of published and in-house methods, and the variety of reference materials and calibrants in use. There was an interest on the part of respondents in validated quantitative assays relevant to all GE events that contain these two genetic elements. Data are presented by testing two variations each of five published real-time quantitative PCR methods for 35S detection on eight maize reference materials. The results showed that two of the five methods were not suitable for all the eight reference materials, with poor linear regression parameters and multiple PCR amplification products for some of the reference materials. This study demonstrates that not all 35S methods produce satisfactory results, emphasizing the need for method validation.

The utility of quantitative molecular diagnostics for patient management depends on the ability to relate patient results to prior results or to absolute values in clinical practice guidelines. To do this, those results need to be comparable across time and methods, either by producing the same value across methods and test versions or by using reliable and stable conversions. Universally available standards and reference materials specific to quantitative molecular technologies are critical to this process but are few in number. This review describes recent history in the establishment of international standards for nucleic acid test development, organizations involved in current efforts, and future issues and initiatives.

The total amount of DNA in a preparation extracted from tissues can be measured in several ways, each method offering advantages and disadvantages. For the sake of accuracy in quantitation, it is of interest to compare these methodologies and determine if good correlation can be achieved between them. Different answers can also be clues to the physical state of the DNA. In this study, we investigated the lack of correlation between ultraviolet (UV) absorbance and fluorescent (PicoGreen) measurements of the concentration of DNAs isolated from plant tissues. We found that quantitation based on the absorbance-based method correlated with quantitation based on phosphorus content, while the PicoGreen-based method did not. We also found evidence of the production of single-stranded DNA under conditions where the DNA was not fragmented into small pieces. The PicoGreen fluorescent signal was dependent on DNA fragment size but only if the DNA was in pure water, while DNA in buffer was much less sensitive. Finally, we document the high sensitivity of the PicoGreen assays to the detergent known as CTAB (cetyldimethylethylammonium bromide). The CTAB-based method is highly popular for low-cost DNA extraction with many published variations for plant and other tissues. The removal of residual CTAB is important for accurate quantitation of DNA using PicoGreen.

An automated sample introduction system, utilizing a demountable direct injection high-efficiency nebulizer (d-DIHEN), is successfully incorporated for the first time with an inductively coupled plasma optical emission spectrometer (ICP-OES) for the measurement of the phosphorus content in acid-digested nucleotides and deoxyribonucleic acid (DNA). With this experimental setup, the solution uptake rate and volume are reduced from 170 to 30 microL min(-1) and from 10 to 2.4 mL, respectively, thereby reducing the required DNA sample mass for solutions containing 3 microg g(-1) P from 300 to 72 microg of DNA, in comparison to previous analyses in our laboratory using a glass concentric nebulizer with cyclonic spray chamber arrangement. The use of direct injection also improves P (I) 213.617 nm sensitivity by a factor of 4 on average. A high-performance (HP) methodology in combination with the previous sample introduction system and ICP-OES provides simultaneous, time-correlated internal standardization and drift correction resulting in relative expanded uncertainties (% U) for the P mass fractions in the range of 0.1-0.4 (95% confidence level) for most of the thymidine 5'-monophosphate (TMP), calf thymus DNA (CTDNA), and plasmid DNA (PLDNA) analyses. The d-DIHEN with HP-ICP-OES methodology allows for the quantification of DNA mass at P mass fractions as low as 0.5 microg g(-1), further reducing the required DNA mass to 12 microg, with small uncertainty (< or = 0.4%). This successful approach will aid in the development and certification of nucleic acid certified reference materials (CRMs), particularly for these samples that are typically limited in volume.

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the flavin adenine dinucleotide (FAD/FADH2) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD+/NADH) and donating them in two separate, one-electron-transfer steps to the iron-sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin-NADP+ reductase (FNR), has no other active redox moieties (e.g., sulfhydryl groups) and can exist in three different oxidation states: (i) oxidized quinone, (ii) one-electron reduced semiquinone (stable neutral species (blue) or unstable radical anion (red)), and (iii) two-electron fully reduced hydroquinone. Here, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of equilibria among the redox components in this initial electron-transfer step of the P450 cycle. A spectroelectrochemical technique was used to measure the midpoint oxidation-reduction potential of PdR that had been carefully purified of all residual NAD+, E0' = -369 +/- 10 mV at pH 7.6, which is more negative than previously reported and more negative than the pyridine nucleotide NADH/NAD+ (-330 mV). After addition of NAD+, the formation of the oxidized reductase-oxidized pyridine nucleotide complex was followed by the two-electron-transfer redox reaction, PdRox:NAD+ + 2e- --> PdRrd:NAD+, when the electrode potential was lowered. The midpoint potential was a hyperbolic function of increasing NAD+ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be E0' = -230 +/- 10 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH. This thermodynamic control of electron transfer is a shared mechanistic feature with the adrenodoxin P450 and photosynthetic electron-transfer systems but is different from the kinetic control mechanisms in the microsomal P450 systems where multiple reaction pathways draw on reducing power held by NADPH-cytochrome P450 reductase. The redox measurements were combined with protein fluorescence quenching of NAD+ binding to oxidized PdR to establish that the PdRox:NAD+ complex (KD = 230 microM) is about 5 orders of magnitude weaker than PdRrd:NAD+ binding. These results are integrated with known structural and kinetic information for PdR, as well as for AdR and FNR, in support of a compulsory ordered pathway to describe the electron-transfer processes catalyzed by all three reductases.

A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated. The reproducibility of the AFLP model was tested by submitting both "unknown" DNA template that had been restricted and ligated with AFLP linkers (R/L mixture) and corresponding primer pairs to nine laboratories participating in the study. Participants completed the final PCR step and then used either slab gel electrophoresis or CE to detect the AFLP fragments. The predicted fragments were identified by the majority of participants with size estimates consistently up to 3 base pair (bp) larger for slab gel electrophoresis than for CE. Shadow fragments, 3 bp larger than the predicted fragments, were often observed by study participants and organizers. The nine AFLP fragments exhibited relative intensities ranging from less than 3% to 22% and, apart from the two weakest fragments, with a % CV of 16 to 25. Fragments containing the highest guanine-cytosine (GC) content of 50-56% showed the greatest stability in the AFLP profiles.

An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.

Measurement of the phosphorus content of nucleotides and deoxyribonucleic acid (DNA) offers an approach to the quantitation of nucleic acids that is traceable to the SI. Such measurements can be an alternative to the commonly used spectroscopic tools that are not traceable. Phosphorus measurements of thymidine 5'-monophosphate (TMP) and acid-digested plasmid and genomic DNA preparations were made using high-performance inductively coupled plasma optical emission spectroscopy (HP-ICP-OES) and high-performance liquid chromatography (HPLC) and compared for bias and uncertainty. A prerequisite for quality measurement is the purity of the materials. Quantitation with the two platforms was comparable for the TMP. However, the HPLC values had larger uncertainties and were all statistically different from the gravimetric values at the 95% confidence level. When using ICP-OES, the digestion of the nucleotide monophosphate can be eliminated, thus simplifying the procedure. The differences between the results obtained by using the two platforms, when measuring genomic or plasmid DNA, were dependent on the mass fraction of the digest. ICP-OES measurement of phosphorus provides a highly accurate quantitation for both nucleotide monophosphates and DNA with expanded uncertainties of less than 0.1%. Currently, ICP-OES requires a significant sample size restricting its usefulness for the quantitation of DNA but represents a valuable tool for certification of reference materials. HPLC requires smaller amounts of material to perform the analysis but is less useful for certification of reference materials because of lower accuracy and 10-fold higher expanded uncertainties.

Objective: This descriptive study examines 45 child and adolescent fatalities related to restraints in residential (institutional) placements in the United States from 1993 to 2003., Method: The study team used common Internet search engines as its primary case discovery strategy to determine the frequency and the nature of the fatalities, as well as the characteristics of the children and the adolescents involved., Results: Male children and adolescents were over-represented in the study sample. Thirty-eight of the fatalities occurred during or after a physical restraint, and 7 fatalities occurred during the use of mechanical restraints. Twenty-eight of the deaths occurred in a prone restraint. In 25 of the fatalities, asphyxia was the cause of death., Conclusion: In the 23 cases in this study where information is available, none of the child behaviors or conditions that prompted the restraint would meet the standard of danger to self or others: the commonly accepted criteria for the use of a restraint. The study points to deficiencies in fatality reporting, recommends reporting fatalities to established state child fatality review boards, and reinforces that restraints be governed by strict protocol and monitoring. The study also urges caution to policymakers in substituting or changing restraint procedures based on the incomplete data reported in this study.

Chorismate lyase (CL) removes the pyruvyl group from chorismate to provide 4-hydroxybenzoate (4HB) for the ubiquinone pathway. We previously reported the crystal structure at 1.4A resolution of the Escherichia coli CL with bound 4HB product, showing that the product is bound in an internal cavity behind two flaps. To provide a more complete basis for understanding CL's unusual ligand-binding properties and mechanism of action, we now report four crystal structures of CL mutants and inhibitor complexes, together with binding and activity measurements and molecular dynamics simulations. First, an ultrahigh resolution (1.0A) crystal structure of the CL*product complex reveals details of a substrate-sized internal cavity, also behind the flaps, near the product site. Second, a 2.4A structure of CL complexed with the inhibitor vanillate shows the flaps partly opened relative to their product-bound positions. Third, a 2.0A structure of the G90A mutant with bound product reveals the basis for tighter product binding and kinetic effects of this active site mutation. Fourth, the combination of the G90A mutation with the vanillate inhibitor produces a 1.9A structure containing two inhibitor molecules, one in the product site and the other in the adjacent cavity. The two sites are connected by a short tunnel that is partly open at each end, suggesting that CL may operate via a 2-site or tunnel mechanism.

We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a conventional molecular beacon. It was found that the overall sensitivities for the four PCR probes are in the order of MB>ATssMB>GCssMB>TaqMan. The fluorescence quantum yield measurements indicate that incomplete or partial enzymatic cleavage catalyzed by Taq polymerase is the likely cause of the low sensitivities of two shared-stem beacons when compared with the conventional beacon probe. A high-fluorescence background associated with the current TaqMan probe sequence contributes to the relatively low detection sensitivity and signal-to-background ratio. The study points out that the nucleotide environment surrounding the reporting fluorophore can strongly affect the probe performance in real-time PCR.

Background: The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results. Statistical analysis of the data arising from three different real-time platforms was conducted in order to assess inter-platform repeatability. On three consecutive days two PCR reaction mixes were used on each of the three amplification platforms - the LightCycler, ABI PRISM 7700 and Rotor Gene 3000. Real-time PCR amplification using a fluorogenic 5' exonuclease assay was performed in triplicate on negative controls and DNA plasmid dilutions of 108-102 copies to give a total of 24 reactions per PCR experiment., Results: The results of the statistical analyses indicated that the platform with the most precise repeatability was the ABI PRISM 7700 when coupled with the FastStart PCR reaction mix. It was also found that there was no obvious relationship between plasmid copy number and repeatability. An ANOVA approach identified the factors that significantly affected the results, in descending order of magnitude, as: plasmid copy number, platform, PCR reaction mix and day (on which the experiment was performed)., Conclusion: In order to deliver useful, informative genetic tests, standardisation of real-time PCR detection platforms to provide repeatable, reliable results is warranted. In addition, a better understanding of inter-assay and intra-assay repeatability is required.

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5' end (T1) is stronger than that labeled at position 5 from the 5' end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5' end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.

The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases. The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.

The structure of the C73G mutant of putidaredoxin (Pdx), the Fe(2)S(2) ferredoxin that supplies electrons to cytochrome CYP101 (p450cam) for camphor oxidation, is reported at 1.9 A resolution in a C2 crystal form. The structure was solved by single-wavelength iron anomalous diffraction, which yielded electron density above the 2sigma level for over 97% of the non-H atoms in the protein. The final structure with R = 0.19 and R(free) = 0.21 has been deposited in the Protein Data Bank with accession code 1r7s. The C2 crystal contains three Pdx molecules in the asymmetric unit, giving three independent models of the protein that are very similar (r.m.s.d. < 0.3 A for the 106 C(alpha) atoms). The unusually high solvent fraction of 80% results in comparatively few crystal-packing artifacts. The structure is briefly compared with the recently reported crystal structures of the C73S and C73S/C85S mutants. In general, the eight independent molecules in the three crystal structures (three in C73G, three in C73S and two in C73S/C85S) are much more similar to each other than to the previously reported NMR structure of wild-type Pdx in solution. The present findings show a unanimous structure in some regions crucial for electron-transfer interactions, including the cluster-binding loop 39-48 and the cytochrome-interaction region of Asp38 and Trp106. In addition, the Cys45 amide group donates a hydrogen bond to cluster sulfur S1, with Ala46 adopting an Lalpha conformation, in all three molecules in the crystal.

Alanine dehydrogenase (AlaDH) from the hyperthermophilic archaeon Archaeoglobus fulgidus is a dimer of 35 kDa chains. The archaeal enzyme appears to represent a new class of AlaDH that is not homologous to bacterial AlaDH enzymes, but has close evolutionary links to the broad ornithine cyclodeaminase/micro-crystallin family, which includes human thyroid hormone binding protein, which has 30% sequence identity to the A. fulgidus gene. The enzyme has been cloned, shown to catalyze the NAD-dependent interconversion of alanine and pyruvate and crystallized in several forms. Although the purified protein crystallized readily under many conditions, most of the crystals diffracted weakly or not at all. One polymorph growing in space group P2(1)2(1)2(1) has non-crystallographic symmetry that becomes crystallographic, changing the space group to P2(1)2(1)2, upon binding iridium or samarium. Before and after derivatization, these crystals diffracted to 2.5 A using synchrotron radiation. Multiwavelength diffraction data were collected from the non-isomorphous iridium derivative, enabling structure determination.

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material requires pure, high-quality genomic DNA as template for subsequent amplification using the polymerase chain reaction (PCR). Six methodologies were evaluated for extracting DNA from ground corn kernels spiked with 0.1% (m/m) CBH351 (StarLink) corn. DNA preparations were evaluated for purity and fragment size. Extraction efficiency was determined. The alcohol dehydrogenase gene (adh1) and the CBH351 (cry9C, 35S promoter) genes in the genomic DNA were detected using PCR. DNA isolated by two of the methods proved unsuitable for performing PCR amplification. All other methods produced some DNA preparations that gave false negative PCR results. We observed that cornstarch, a primary component of corn kernels, was not an inhibitor of PCR, while acidic polysaccharides were. Our data suggest that amplification of an endogenous positive control gene, as an indicator for the absence of PCR inhibitors, is not always valid. This study points out aspects of DNA isolation that need to be considered when choosing a method for a particular plant/tissue type.

In Petunia inflata, a species with gametophytic self-incompatibility, pollination triggers two phases of ethylene production by the pistil, the first of which peaks 3 hours after pollination with compatible or incompatible pollen. To investigate the physiological significance of the first phase of ethylene production, pollinated flowers were treated with 2,5-norbornadiene (NBD), an inhibitor of ethylene action. Treatment with NBD reduced pollen tube growth in a dose-dependent manner during the first six hours after pollination; however, pollen tube growth was insensitive to NBD if the treatment was applied 6 hours or more after pollination. Simultaneous application of exogenous ethylene substantially offset the inhibitory effects of NBD in flowers pollinated for 4 hours. Another inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), also produced a strong inhibition of pollen tube growth during the first six hours of pollination. The experiments with 1-MCP pretreatment indicate that pistil tissues are the primary target of the pollination-induced ethylene.

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR., (Copyright 2003 Wiley Periodicals, Inc.)

Spectroelectrochemistry measurements are used to demonstrate that active site mutation and binding of an non-natural substrate to P450cam (CYP101) reduces the shift in the redox potential caused by substrate-binding, and thereby results in slower catalytic turnover rate relative to wild-type enzyme with the natural camphor substrate.

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4-hydroxy benzoate (4HB) for the ubiquinone pathway. In Escherichia coli, CL is monomeric, with 164 residues. We have determined the structure of the CL product complex by crystallographic heavy-atom methods and report the structure at 1.4-A resolution for a fully active double Cys-to-Ser mutant and at 2.0-A resolution for the wild-type. The fold involves a 6-stranded antiparallel beta-sheet with no spanning helices and novel connectivity. The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main-chain amides and by the buried side-chains of Arg 76 and Glu 155. The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix-turn-helix loops. The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10-fold stronger binding of product than substrate. Substrate binding and kinetically rate-limiting product release apparently require the rearrangement of these active-site-covering loops. Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane-bound enzyme 4HB octaprenyltransferase.

Putidaredoxin (Pdx), a [2Fe-2S] redox protein of size M(r) 11,600, transfers two electrons in two separate steps from the flavin containing putidaredoxin reductase to the heme protein, cytochrome CYP101 in the P450cam catalytic cycle. It has recently come to light, through NMR measurements, that there can be appreciable differences in the Pdx conformational dynamics between its reduced and oxidized states. The redox reaction entropy, deltaS(0')rc = (S(0')Pdx(r)-S(0')Pdx(0)), as determined from measurements of the variation in formal potential with temperature, E0'(T), provides a measure of the strength of this influence on Pdx function. We designed a spectroelectrochemical cell using optically transparent tin oxide electrodes, without fixed or diffusible mediators, to measure E0'(T) over the temperature range 0-40 degrees C. The results indicate that the redox reaction entropy for Pdx is biphasic, decreasing from -213 +/- 27 J mol(-1) K(-1) over 0-27 degrees C, to -582 +/- 150 J mol(-1) K (-1) over 27-40 degrees C. These redox reaction entropy changes are significantly more negative than the changes reported for most cytochromes, although our measurement over the temperature interval 0-27 degrees C is in the range reported for other iron-sulfur proteins. This suggests that Pdx (and other ferredoxins) is a less rigid system than monohemes, and that redox-linked changes in conformation, and/or conformational dynamics, impart to these proteins the ability to interact with a number of redox partners.

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(aq) = anthranilate(aq) + pyruvate(aq) + H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq) + ammonia(aq) = ADIC(aq) + H2O(l) and ADIC(aq) = anthranilate(aq) + pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate2-(aq) + NH4+(aq) = anthranilate-(aq) + pyruvate-(aq) + H+(aq) + H2O(l), delta rHmo = -(116.3 +/- 5.4) kJ mol-1. For the reaction: chorismate2-(aq) + NH4+(aq) = ADIC-(aq) + H2O(l), K = (20.3 +/- 4.5) and delta rHmo = (7.5 +/- 0.6) kJ mol-1. Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes delta rG'mo under approximately physiological conditions are given.

Chorismate pathway enzymes are important as producers of nonnucleotide aromatic compounds. The enzyme chorismate lyase from Escherichia coli has been crystallized in four distinct forms, three of which have been characterized by X-ray diffraction. Despite widespread screening, all four crystal forms grow from the same chemical conditions. The wild-type enzyme tends to aggregate, even in the presence of reducing agent, and yielded only one crystal form (monoclinic, form 1) that grew in intricate clusters. Chemical modification of the cysteines mitigated problems with aggregation and solubility but did not affect crystal growth behavior. Protein aggregation was largely eliminated by mutating the protein's two cysteines to serines. The double mutant retains full enzymatic activity and crystallizes in three new forms, one of which (triclinic) diffracts to 1.1-A resolution., (Copyright 2000 Academic Press.)

The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T1 and T2 values and 1H-15N NOEs have been measured for the diamagnetic region of the protein. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameters (S2), the effective correlation time for internal motions (tau e), and the 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (tau(m)). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on the microsecond to millisecond time scale for the oxidized Pdx, in comparison with the reduced Pdx. These results clearly indicate that the oxidized protein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proton exchange. In addition, we observed very high T1/T2 ratios for residues 33 and 34, giving rise to a large Rex contribution. Residue 34 is believed to be involved in the binding of Pdx to cytochrome P450cam (CYP101). The differences in the backbone dynamics are discussed in relation to the oxidation states of Pdx, and their impact on electron transfer. The entropy change occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.

Putidaredoxin is a di-iron protein whose paramagnetic region is not well characterized by 1H detected NMR. We have studied the structure of this region in greater detail by directly observed 15N NMR of oxidized and reduced putidaredoxin preparations in which the six cysteine residues are selectively labeled with 15N. A new method for preparation of a stable form of reduced putidaredoxin has been developed for use in NMR. The 15N NMR spectra of the oxidized and reduced forms are characteristically different, and we have measured and compared 15N chemical shifts, spin-lattice relaxation times (T1), and chemical shift/temperature dependences for both forms. Evidence for localized valencies of the iron atoms in the reduced form is presented. From the 15N T1 values of the oxidized form, reduced distances of the cysteine backbone 15N nuclei from the center of the Fe2S2 cluster have been calculated. These distances are consistent with those calculated from X-ray crystal structure data for five ferredoxins, and confirm the structural similarity of the Fe2S2 clusters in putidaredoxin and in these ferredoxins in the oxidized state.

Reduction of Cu2+ by plasma membrane vesicles isolated from tomato (Lycopersicon esculentum Mill.) roots was investigated. Plants were grown in hydroponic culture with complete nutrition for 4 weeks or were deprived of Fe for the last 7 d. Plasma membrane vesicles were prepared by aqueous two-phase partitioning. Reduction of Cu, Fe, and ferricyanide by plasma membrane vesicles was measured. An increase in the activity of all three pyridine-nucleotide-dependent activities was noted in plasma membrane preparations from Fe-deficient, compared to Fe-sufficient, plants. Solubilization and chromatographic separation of two plasma membrane electron transport systems indicated that the Fe-chelate reductase was probably responsible for reduction of Cu. Assays used a variety of Cu chelates, and for each the Cu activity in the assay was determined by the program Geochem PC. The rate of reduction of Cu correlated with the level of Cu activity, and results support the idea that free Cu2+ and not Cu chelates may serve as the true substrate for reduction. Reduction was observed only in assays in which Cu activity was equivalent to Cu-enriched or Cu-toxic soils. These results suggest that reduction of Cu by tomato root may have little or no physiological relevance under conditions experienced by the root in the soil.

The mitochondrial outer membrane channel, VDAC, provides a pathway for the flux of metabolites between the cytoplasm and mitochondrion. VDAC is voltage-dependent and occupies states of differing conductivity and ion selectivity that are dependent on transmembrane potential. A protein, derived from preparations of mitochondria, has been shown to increase the voltage dependence of VDAC and is called the VDAC modulator. Both VDAC and the VDAC modulator have been extensively characterized by reconstitution into planar lipid bilayers. In order for the VDAC modulator to have physiological significance it must have physical access to VDAC in the cell. This constraint dictates that the modulator be an extrinsic outer mitochondrial membrane protein, occupy the mitochondrial intermembrane space, or be a cytoplasmic constituent. To address the question of subcellular localization, purified mitochondria were selectively lysed with digitonin or treated with trypsin while resuspended in hypo-osmotic or iso-osmotic medium. Marker enzymes and modulator activity were monitored during the various treatments. Results indicate that the integrity of the outer membrane was necessary to prevent modulator release or protection from trypsin digestion. Outer membrane lysis, under conditions where the inner membrane remained intact, resulted in modulator release or inactivation by trypsin. These results suggest an intermembrane space location for the VDAC modulator in the mitochondrion.

Reduction of Fe(3+) to Fe(2+) is a prerequisite for Fe uptake by tomato roots. Ferric chelate reductase activity in plasma membranes (PM) isolated from roots of both iron-sufficient (+Fe) and iron-deficient (-Fe) tomatoes (Lycopersicon esculentum Mill.) was measured as NADH-dependent ferric citrate reductase and exhibited simple Michaelis-Menten kinetics for the substrates, NADH and Fe(3+)(citrate(3-))(2). NADH and Fe(3+)(citrate(3-))(2)K(m) values for reductase in PM from +Fe and -Fe tomato roots were similar, whereas V(max) values were two- to threefold higher for reductase from -Fe tomatoes. The pH optimum for Fe-chelate reductase was 6.5. Fe-chelate reductases from -Fe and +Fe tomato roots were equally sensitive to several triazine dyes. Reductase was solubilized with n-octyl beta-d-glucopyranoside and electrophoresed in nondenaturing isoelectric focusing gels. Three bands, with isoelectric points of 5.5 to 6.2, were resolved by enzyme activity staining of electrofocused PM proteins isolated from +Fe and -Fe tomato roots. Activity staining was particularly enhanced in the isoelectric point 5.5 and 6.2 bands solubilized from -Fe PM. We conclude that PM from roots of +Fe and -Fe plants contain Fe-chelate reductases with similar characteristics. The response to iron deficiency stress likely involves increased expression of constitutive Fe-chelate reductase isoforms in expanding epidermal root PM.

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Deltapsi) could also be detected indirectly as (45)Ca(2+) uptake. Hmt toxin added to T mitochondria dissipated the steady state Deltapsi similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C(41)H(68)O(12) and Cpd IV: C(25)H(44)O(6)), reduced native toxin (RT2C: C(41)H(84)O(13)) and Pm toxin (band A: C(33)H(60)O(8), produced by the fungus, Phyllosticta maydis) were effective in dissipating Deltapsi and decreasing Ca(2+) uptake with the following order: Pm (100) >> HmT (23-30) > Cpd XIII (11-25) >> RT2C (0-4-1.8) > Cpd IV (0.2-1.0). In contrast, the toxins and analogs had no effect on Deltapsi formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C(41)H(68)O(13)) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Deltapsi in energized mitochondria is caused mainly by the electrogenic extrusion of H(+), the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H(+). The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H(+) leakage.

The mitochondrial outer membrane channel, VDAC, serves as the primary permeability pathway for metabolite flux between cytoplasmic and mitochondrial compartments. VDAC can occupy several conformational states differing in ion conductivity. Small transmembrane potentials cause transitions from open- to closed-channel conformations. A soluble mitochondrial protein enhances the channel's response to voltage by increasing the rate of channel closing; inducing the occupation of lower conductance states; and decreasing the rate of channel reopening. This protein modulator acts at very low concentrations and its role in the cell may be to regulate the permeability of the mitochondrial outer membrane by inducing channel closure.

The mitochondrial outer membrane contains voltage-gated channels called VDAC that are responsible for the flux of metabolic substrates and metal ions across this membrane. The addition of micromolar quantities of aluminum chloride to phospholipid membranes containing VDAC channels greatly inhibits the voltage dependence of the channels' permeability. The channels remain in their high conducting (open) state even at high membrane potentials. An analysis of the change in the voltage-dependence parameters revealed that the steepness of the voltage dependence decreased while the voltage needed to close half the channels increased. The energy difference between the open and closed states in the absence of an applied potential did not change. Therefore, the results are consistent with aluminum neutralizing the voltage sensor of the channel. pH shift experiments showed that positively charged aluminum species in solution were not involved. The active form was identified as being either (or both) the aluminum hydroxide or the tetrahydroxoaluminate form. Both of these could reasonably be expected to neutralize a positively charged voltage sensor. Aluminum had no detectable effect on either single-channel conductance or selectivity, indicating that the sensor is probably not located in the channel proper and is distinct from the selectivity filter.

The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca(2+) gradients, induced by the toxin in T corn was prevented by N,N'-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N'-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site.

Though Helminthosporium maydis race T (HmT) toxin decreased active Ca(2+) uptake into mitochondria isolated from susceptible (T) but not resistant (N) corn (Kimber, Sze, 1984 Plant Physiol 74: 804-809 the mode of toxin action is not understood. This study shows that HmT toxin or A23187 (a Ca(2+) ionophore) dissipated a Ca(2+) gradient in T mitochondria. However, HmT toxin had no effect on Ca(2+) gradients in N mitochondria or microsomal vesicles from T or N corn. The results suggest that HmT toxin increased membrane permeability to Ca(2+) in mitochondria of T corn specifically.

It has recently been suggested that following the exposure of root surface collagen with citric acid, the addition of topically applied fibronectin might promote healing with a fibrous re-attachment. The purpose of this study was to determine the benefit of citric acid demineralization and fibronectin application in the surgical treatment of severe, naturally occurring periodontal disease in Beagle dogs. The 4 treatment modalities employed were: (1) surgery alone (mucoperiosteal flaps); (2) surgery plus fibronectin; (3) surgery plus citric acid; (4) surgery plus citric acid followed by fibronectin application. Coronal and root surface notches were used as biometric and histometric reference points. Final clinical measurements were recorded 6 weeks post surgically, on the day of sacrifice. Significantly increased amounts of connective tissue reattachment were observed in the areas treated with the citric acid/fibronectin combination. Fibrous re-attachment was enhanced at the expense of epithelial downgrowth and occurred directly to both new and old cementum and exposed dentin, often in a functional manner, i.e., perpendicular to the root surface. Areas treated with the surgery and citric acid technique attained moderate amounts of fibrous re-attachment while the other treatment modalities were associated with a long junctional epithelium. The enhanced fibrous re-attachment may be the product of an accelerated coalescing of exposed soft tissue and root surface collagen fibrils, while under the mediating effect of fibronectin.