Test of CAP88-PC's predicted concentrations of tritium in air at Lawrence Livermore National Laboratory.

Based on annual tritium release rates from the five sources of tritium at Lawrence Livermore National Laboratory and the Tritium Research Laboratory at Sandia National Laboratory, the regulatory dispersion and dose model, CAP88-PC, was used to predict tritium concentrations in air at perimeter and offsite air surveillance monitoring locations for 1986 through 2001. These predictions were compared with mean annual measured concentrations, based on biweekly sampling. Deterministic predictions were compared with deterministic observations using predicted-to-observed ratios. In addition, the uncertainty on observations and predictions was assessed: when the uncertainty bounds of the observations overlapped with the uncertainty bounds of the predictions, the predictions were assumed to agree with the observations with high probability. Deterministically, 54% of all predictions were higher than the observations, and 96% fell within a factor of three. Accounting for uncertainty, 75% of all predictions agreed with the observations; 87% of the predictions either matched or exceeded the observations. Predictions equaled or exceeded observations at those sampling locations towards which the wind blows most frequently, except those in the hills. Under-predictions were seen at locations towards which the wind blows infrequently when released tritium was from elevated sources. When a high fraction of tritium was from area (diffuse) sources, predictions matched observations.

Tritium doses from chronic atmospheric releases: a new approach proposed for regulatory compliance.

Regulatory models for atmospheric releases of tritium approved by the Environmental Protection Agency (CAP88, AIRDOS-PC, and COMPLY) calculate doses only from tritiated water (HTO) taken into the body. They do not deal with dose from emissions of tritiated hydrogen gas (HT) and conversion of HT to HTO in the environment, nor do they address the dose from ingesting tritium incorporated into organic compounds. A simple model (NEWTRIT) is proposed that accounts for all pathways to dose from atmospheric releases of HT and HTO. The model is formulated in terms of the tritium-to-hydrogen ratio in each environmental compartment. With each transfer, a small reduction in the ratio is introduced to reflect the dilution that occurs in nature. Conversion of HT to HTO in the environment is modeled using the latest experimental data. Concentrations of organically bound tritium are calculated in foodstuffs based on amounts of hydrogen in proteins, fats, and carbohydrates. Concentrations in foodstuffs and doses calculated by NEWTRIT are consistent with the predictions of existing regulatory models. In addition, the HTO component of NEWTRIT is tested using public bioassay data and the HT component is tested using results from a model intercomparison study for a hypothetical HT release. Although tritium doses probably have not been underestimated by regulatory models that account only for HTO (due to the high degree of conservatism built into these models), the explicit treatment of HT and organically bound tritium proposed here will make the dose assessments more comprehensive, defensible, and scientifically acceptable. Because NEWTRIT includes all pathways to dose and predicts conservative doses, it is a suitable model to replace the tritium models currently used for compliance.

Inherited bleeding disorders in dermatologic surgery.

BACKGROUND: A patient referred for Mohs micrographic surgery of a basal cell carcinoma had a history of a congenital clotting factor IX deficiency requiring recombinant factor IX replacement. OBJECTIVE: To examine the management and problems associated with cutaneous surgery in patients with inherited clotting factor deficiencies. METHODS: Case report and review of the medical literature. RESULTS: Reconstructive options must be carefully chosen to minimize bleeding in patients with clotting factor deficiencies. Preoperative consultation with a hematologist and coagulation factor replacement both before and after cutaneous surgery prevent excessive hemorrhage. CONCLUSION: Meticulous attention to hemostasis, careful preoperative assessment, and postoperative follow-up minimize complications in patients with known coagulation deficiencies who require cutaneous surgery.

Influence of gender differences in the carbon pool on dose factors for intakes of tritium and 14C-labeled compounds.

The ICRP's biokinetic models for five tritium-labeled and five 14C-labeled compounds (not including radiopharmaceutical compounds and excepting carbon monoxide) incorporate a compartment representing the body carbon pool. Using the ICRP models, as coded into the Genmod-PC internal dosimetry code, higher dose coefficients are calculated for females than for ICRP's Reference Man. The ICRP's committed effective dose coefficients for the ingestion of tritiated water and organically bound tritium by the adult male are 1.8 x 10(-11) and 4.2 x 10(-11) Sv Bq(-1), respectively. Using the Genmod-PC code, the corresponding dose coefficients for the adult female are 2.2 x 10(-11) and 6.2 x 10(-11) Sv Bq(-1), which are 25% and 46% greater than the adult male's. Similarly, the ICRP's dose coefficient is 5.8 x 10(-10) Sv Bq(-11) for an intake of organically bound 14C by the adult male, and the estimated dose coefficient using Genmod is 54% greater for the adult female. The carbon retention half-time for an average adult female is calculated as 51 d and that for an average adult male, 38 d; the latter is similar to the carbon half-time of 40 d recommended by International Commission on Radiological Protection (ICRP). The longer turnover time of whole body carbon in females is one factor that causes the dose coefficients for females to be higher than those of males; a second factor is the smaller whole body mass of ICRP's Reference Woman compared to Reference Man.

A metabolic derivation of tritium transfer coefficients in animal products.

Tritium is a potentially important environmental contaminant originating from the nuclear industry, and its behaviour in the environment is controlled by that of hydrogen. Animal food products represent a potentially important source of tritium in the human diet and a number of transfer coefficient values for tritium transfer to a limited number of animal products are available. In this paper we present an approach for the derivation of tritium transfer coefficients which is based on the metabolism of hydrogen in animals. The derived transfer coefficients separately account for transfer to and from free (i.e. water) and organically bound tritium. A novel aspect of the approach is that tritium transfer can be predicted for any animal product for which the required metabolic input parameters are available. The predicted transfer coefficients are compared to available independent data. Agreement is good (R2=0.97) with the exception of the transfer coefficient for transfer from tritiated water to organically bound tritium in ruminants. This may be attributable to the particular characteristics of ruminant digestion. We show that tritium transfer coefficients will vary in response to the metabolic status of an animal (e.g. stage of lactation, diet digestibility etc.) and that the use of a single transfer coefficient from diet to animal product is inappropriate. It is possible to derive concentration ratio values from the estimated transfer coefficients which relate the concentration of tritiated water and organically bound tritium in an animal product to their respective concentrations in the animals diet. These concentration ratios are shown to be less subject to metabolic variation and may be more useful radioecological parameters than transfer coefficients. For tritiated water the concentration ratio shows little variation between animal products ranging from 0.59 to 0.82. In the case of organically bound tritium the concentration ratios vary between animal products from 0.15 (goat milk) to 0.67 (eggs).

Digitally collected cryo-electron micrographs for single particle reconstruction.

Several advantages and disadvantages have been cited for image collection with a slow-scan CCD camera. Here we explore its use for cryo-EM single particle reconstruction and present two practical examples. The icosahedral adenovirus (Ad) type 2 ( approximately 150 MDa) was reconstructed from 396 particle images. The Fourier shell correlation (FSC) 0.5 threshold and the Fourier shell phase residual (FSPR) 45 degrees criterion yielded 17 AA resolution for the ordered viral capsid. Visual comparison with the filtered Ad2 crystallographic hexon confirmed a resolution range of 15-17 A. The asymmetric DNA-PKcs protein (470 kDa) was reconstructed from 9,473 particle images, using a previously published reconstruction based on class-sum images as an orientational search model [Chiu et al. (1998) J. Mol. Biol. 284:1075-1081]. FSC and FSPR methods yielded 17 A resolution for the new DNA-PKcs reconstruction, indicating a small but noticeable improvement over that of the class-sum based reconstruction. Despite the lack of symmetry for DNA-PKcs and its lower image contrast compared to Ad2 (0.8% vs. 2.5%), the same resolution was obtained for both particles by averaging significantly more DNA-PKcs images. Use of the CCD camera enables the microscopist to adjust the electron beam strength interactively and thereby maximize the image contrast for beam sensitive samples. On-line Fourier transformation also allows routine monitoring of drift and astigmatism during image collection, resulting in a high percentage of micrographs suitable for image processing. In conclusion, our results show that digital image collection with the YAG-scintillator slow-scan CCD camera is a viable approach for 3D reconstruction of both symmetric and asymmetric particles.

Modeled concentrations in rice and ingestion doses from chronic atmospheric releases of tritium.

The expansion of nuclear power programs in Asia has stimulated interest in the improved modeling of concentrations of tritium in rice, a staple crop grown throughout the far east. Normally, the specific activity model is used to calculate concentrations of tritium in the tissue water of edible plants to assess ingestion dose from chronic releases. However, because rice, like other grains, has much lower water content than most crops, the calculation must also account for organically bound tritium. This paper reviews ways to calculate steady-state concentrations of tritium in rice, including the methods of Canadian and United States regulatory models, and the assumptions behind them. Concentrations in rice and resulting ingestion doses are compared for the various methods, and equations for calculating concentrations are recommended. The regulatory models underestimate doses received from ingestion of rice contaminated with tritium since they do not account explicitly for organically bound tritium. The importance of including organically bound tritium is illustrated in a comparison of doses from rice, leafy vegetables and milk for an Asian diet. Dose factors from tritium for these foods are estimated to be 135, 47, and 20 nSv y(-1)/(Bq m(-3)), respectively. Assuming known air concentrations, tritium concentrations in rice, calculated with the recommended equations, are uncertain by less than a factor 2 when tritium concentrations in the rice paddy water are known, and by less than a factor of 2.3 when concentrations in paddy water are unknown.

Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors.

The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.

Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.

The solution structure of the DNA double-stranded break repair protein Ku and its complex with DNA: a neutron contrast variation study.

Small-angle X-ray and neutron scattering with contrast variation has been used to study the structure of the DNA targeting component (Ku) of the DNA-dependent protein kinase and its complex with DNA. The Ku protein in solution has the approximate shape of a prolate ellipsoid with semi-axes of 24, 43, and 89 A. In the presence of a minimal-length DNA binding sequence (a 24-base-pair duplex DNA), a 1:1 Ku/DNA complex forms. This 1:1 stoichiometry is observed when either the Ku or the DNA is in excess. Analysis of the contrast variation data on Ku complexed with either the 24-mer duplex DNA or a slightly longer 30-mer duplex DNA shows that both the DNA and Ku structures have the same overall conformations within the 1:1 complex as the uncomplexed components. The separation of the centers-of-mass for the Ku/24-mer DNA complex is 46 A, while that for the Ku/30-mer DNA is 56 A. The DNA binds within what appears to be a preformed channel that penetrates deeply into the Ku protein such that the entire length of the 24-mer DNA spans the protein. The slightly longer 30-mer binds in a similar fashion, but with its extra length protruding from the protein envelop. The scattering data are consistent with the idea that the Ku "threads" onto the duplex DNA via a channel that can completely bury approximately 24 base pairs.

Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK) plays an important role in mammalian DNA double-strand break repair and immunoglobulin gene rearrangement. The DNA-PK holoenzyme is activated by assembly at DNA ends and is comprised of DNA-PKcs, a 460 kDa protein kinase catalytic subunit, and Ku, a 70 kDa/80 kDa heterodimeric DNA-targeting component. We have solved the three-dimensional structure of DNA-PKcs to approximately 21 A resolution by analytically combining images of nearly 9500 individual particles extracted from cryo-electron micrographs. The DNA-PKcs protein has an open, pseudo 2-fold symmetric structure with a gap separating a crown-shaped top from a rounded base. Columns of density are observed to protrude into the gap from both the crown and the base. Measurements of the enclosed volume indicate that the interior of the protein is largely hollow. The structure of DNA-PKcs suggests that its association with DNA may involve the internalization of double-stranded ends.

A nondenaturing purification scheme for the DNA-binding domain of poly(ADP-ribose) polymerase, a structure-specific DNA-binding protein.

Poly(ADP-ribose) polymerase (PARP) is thought to be involved in DNA repair given its ability to recognize and bind to DNA strand breaks. During apoptosis, PARP is proteolytically cleaved into two stable fragments, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa fragment containing the automodification and catalytic domains. To understand the DNA-binding properties of PARP, we expressed a recombinant hexahistidine tagged protein (His-DBD) in Escherichia coli. We modified expression to facilitate protein folding by including zinc and reducing the induction temperature. Properly folded, the DNA-binding domain of PARP binds to single- and double-stranded DNA in a structure-specific manner. To eliminate contamination with bacterial DNA that occurred during the purification process, a purification procedure was developed to produce DNA-free protein. In addition, to remove the hexahistidine tag from the recombinant protein, thrombin cleavage was carried out while the recombinant protein was bound to a DNA column. This procedure stabilized the recombinant protein and resulted in nearly 100% cleavage with no appreciable loss to unwanted proteolytic degradation. This nondenaturing purification scheme results in high-quality, native PARP-DBD for use in structural and biochemical studies.

Antinuclear antibody seropositivity in patients with cutaneous T-cell lymphoma.

BACKGROUND: We attempted to determine the frequency and clinical relevance of antinuclear antibody (ANA) testing and positive ANA test results in patients with cutaneous T-cell lymphoma (CTCL). METHODS: A retrospective chart and computer record review was conducted to determine the frequency of ANA testing in CTCL patients and the rate of seropositivity. Patients with a positive ANA were further examined to define possible explanations of the positive test. RESULTS: Of 381 patients with CTCL, 66 (17%) had ANA tests; 8 of these (12.1%) were found to have an ANA titer greater than or equal to 1:40. Of patients with a positive ANA test, one was found to have chronic cutaneous lupus erythematosus histologically and clinically mimicking CTCL. Others were found to have a comorbid connective tissue disorder, some had apparent drug-induced antinuclear antibodies, and some had no identifiable reason for a positive ANA test. CONCLUSION: ANA seropositivity does not appear to be increased in CTCL patients, and the ANA test remains a useful screening tool for differentiating between CTCL and connective tissue disorders.

Dermatologists meet the primary care standard for first contact management of skin disease.

BACKGROUND: It has been suggested that first contact for skin disease should be the domain of primary care providers because they provide comprehensive services beyond those offered by dermatologists. OBJECTIVE: The purpose of this study was to test the hypothesis that dermatologists do not meet the standards for providing primary care to which generalists are held. METHODS: National Ambulatory Medical Care Survey data from the year 1995 were used to determine the frequency at which counseling and preventive examinations were performed at visits to primary care providers and dermatologists. RESULTS: Counseling and preventive examinations were performed at a minority of visits for skin disease. No counseling was reported at 91% of the visits to primary care providers and at 94% of visits to dermatologists. Preventive examinations other than blood pressure were done at 4.7% of the visits to primary care providers and at 1.5% of visits to dermatologists. CONCLUSION: The standard of primary care for skin disease, as set by the generalist, is attention to the skin disease and not comprehensive medical care. Dermatologists are best able to meet this standard.

Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase.

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.

Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints.

We investigated the requirements for protein p53 and the ATM gene product in radiation-induced inhibition of DNA synthesis and regulation of the cyclin E/ and cyclin A/cyclin dependent kinases (Cdks). Wild type (WT) mouse lung fibroblasts (MLFs), p53(-/-) knock-out MLFs, normal human skin fibroblasts (HSF-55), and human AT skin fibroblasts (GM02052) were used in the investigations. The absence of p53 had no significant effect on the inhibition or recovery of DNA synthesis throughout the S phase, as determined from BrdU labeling and flow cytometry, or the rapid inhibition of cyclin A/Cdks. Gamma radiation (8 Gy) inhibited DNA synthesis and progression into G2 during the first 3 h after irradiation, and the recovery of these processes occurred at similar rates in both WT and p53(-/-) MLFs. The cyclin A/Cdks were inhibited 55-70% at 1 h after irradiation in both cell types, but p21WAF1/Cip1 levels or p21 interaction with Cdk2 did not increase in the irradiated p53(-/-) MLFs. Although p53(-/-) MLFs do not exhibit prolonged arrest at a G1 checkpoint, radiation did induce a rapid 20% reduction and small super-recovery of cyclin E/Cdk2 within 1-2 h after irradiation. Similar inhibition and recovery of cyclin E/Cdk2 previously had been associated with regulation of transient G1 delay and the inhibition of initiation at an apparent G1/S checkpoint in Chinese hamster cells. In contrast, loss of the ATM gene product abrogated transient cyclin E/Cdk2 inhibition, most inhibition of DNA synthesis and all, but a 10-15% inhibition, of the cyclin A/Cdks. The results indicate that neither p53 nor p21 is required for transient inhibition of cyclin E/Cdk2 associated with the G1/S checkpoint or for inhibition of DNA synthesis at 'checkpoints' within the S phase. Conversely, the ATM gene product appears to be essential for regulation of the G1/S checkpoint and for inhibition of DNA replication associated with the inhibition of cyclin A/Cdk2. Differential aspects of DNA synthesis inhibition among cell types are presented and discussed in the context of S phase checkpoints.

Data quality and validation of radiological assessment models.

Simulation models must be tested against measurements before their predictions can be viewed as reliable. However, having data to test all pathways in a model is rare, and finding data that were collected for the purpose of model testing is even rarer. Thus modelers must take every opportunity to test portions of their models with data obtained under less than ideal circumstances. With careful preparation of the data and a thorough understanding of the data requirements of the model, the success of the test can be assured. Monitoring data collected after the Chernobyl reactor accident were used for model testing in two international studies, the Biospheric Model Validation Study and the Validation of Environmental Model Predictions. Based on these experiences, this paper describes the data requirements of validation, the validation of models with less than ideal data, the value of good data, the role of modeling in improving the quality of data and, most importantly, ways to improve collection and preparation of data to assist further validation. Although the examples are specific to radiological assessment models, the principles of data collection and preparation herein may be applied to testing all simulation models.

Ku80 gene expression is Sp1-dependent and sensitive to CpG methylation within a novel cis element.

The Ku70/80 complex, known as Ku, constitutes the DNA end binding component of the DNA-dependent protein kinase (DNA-PK). We have characterized the promoter region of the mouse and human Ku80 genes to delineate transcriptional elements necessary for basal gene expression and proliferation-dependent regulation. Consensus Sp1 recognition elements were identified in both promoters, and were determined to be essential for basal expression. We further identified a near-perfect palindrome of 21 base pairs located immediately 5' to one Sp1 element. This sequence was present once within the mouse Ku80 promoter and seven times, in a head-to-tail tandem array, within the human Ku80 promoter. This sequence possessed homology with a methylation-sensitive promoter element, Enh2, present in the LTR of mouse intractisternal A-particles. Promoter deletion studies and expression analysis of in-vitro methylated reporter gene constructs provided strong evidence that, in vivo, this repeat sequence regulates Ku80 gene expression in cis, through a mechanism involving CpG methylation. Evidence is also presented, suggesting that Ku is directly involved in this regulatory process.

DNA looping by Ku and the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an approximately 460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein-nucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.