Megavoltage bremsstrahlung end point voltage diagnostic.

In a material, a beam of x rays is accompanied by various kinds of secondary radiation, including Compton electrons from collisions between the x rays and the material's electrons. For megavoltage bremsstrahlung in air, many of these Compton electrons are forward-directed and fast enough to be deflected outside the beam's edge by a magnetic field perpendicular to the beam. At the beam's edge, the dose from the deflected Compton electrons has a pattern that depends on the radiation's end point energy. Dose patterns measured with radiochromic film on a nominally 1 and 2 MV linear accelerator agree reasonably well with the corresponding Monte Carlo computations. With further development, the dose pattern produced outside the beam by such a sweeper magnet could become a noninvasive way to monitor megavoltage bremsstrahlung, when the end point energies are difficult to determine with other methods.

Comparison of parental reports of smoking and residential air nicotine concentrations in children.

BACKGROUND: Using questionnaires to assess children's residential exposure to environmental tobacco smoke (ETS) may result in misclassification from recall and response bias. Questionnaire data have frequently been validated against urinary cotinine measurements, but rarely against actual measurements of residential air nicotine. OBJECTIVE: To compare questionnaire reported smoking with air nicotine concentrations in a large population of children and with urinary cotinine levels in a subpopulation; and to assess the potential impact of the symptom status of the children on the agreement between different measures of exposure. METHODS: The authors assessed residential exposure to ETS in 347 German, 335 Dutch, and 354 Swedish preschool and schoolchildren by questionnaire and air nicotine measurements, and in a subset of 307 German children by urinary cotinine measurements. They then compared the different measures of ETS exposure. RESULTS: In all countries, air nicotine concentrations increased with increasing questionnaire reported smoking in a dose-response fashion. Specificity and negative predictive values of questionnaire reports for nicotine concentrations were excellent. Sensitivity and positive predictive values were moderate to good. Excluding occasional smokers, the overall percentage of homes misclassified was 6.9%, 6.7%, and 5.1% in Germany, the Netherlands, and Sweden, respectively. Similar results were found for the agreement of urinary cotinine concentrations with questionnaire reports and air nicotine levels. There was no indication of underreporting by parents of symptomatic children. CONCLUSION: Despite some misclassification, questionnaire reports are an inexpensive and valid estimate of residential ETS exposure among preschool and school children.

Biomarkers of exposure to passive smoking of school children: frequency and determinants.

UNLABELLED: This study aims to assess the extent of children' exposure to ETS and quantify potential determinants. A total of 2767 children aged 5-14 years participated in an environmental survey in East Germany in 1998-1999 (participation rate 75.9%). A subgroup of 979 children between the ages of 11 and 14 years with complete data on nicotine and cotinine in urine were selected for this analysis. This study population consisted of 73 self-reported smokers (7.5%), 793 non-smokers (81%) and 113 children with missing data on smoking status (11.5%). Nicotine and cotinine concentrations in spontaneous urine sample were determined by high-performance liquid chromatography methods with ultraviolet-detection and corrected for creatinine. Approximately 40% of self-reported non-smokers were exposed to environmental tobacco smoke (ETS) at home. Non-smoking children exposed to parental tobacco smoke at home compared with not exposed showed in average higher nicotine and cotinine concentration (geometric mean 4.7 microg/l vs. 1.4 microg/l and 8.1 microg/l vs. 2.7 microg/l) and the adjusted odds ratio (OR) for detectable biomarkers ranged between 17 and 22. There were increased rates of detectable biomarkers in urine with increasing numbers of smoked cigarettes in the household (adjusted OR increased from 8 to 54). Maternal smoking showed a stronger effect than paternal smoking. Furthermore, low parental education, cold season, height of dwelling (

Characterization of a naphthalene derivative inhibitor of retroviral integrases.

The retroviral integrase protein (IN) is essential for virus replication and, therefore, an attractive target for the development of inhibitors to treat human immunodeficiency virus (HIV) infection. Diverse classes of compounds that are active against this protein have been discovered using in vitro assays. Here we describe the synthesis of a novel compound, 3,8-dibromo-7-amino-4-hydroxy-2-naphthalenesulfonic acid (2BrNSA), which inhibits the in vitro activities of the full-length HIV-1 and avian sarcoma virus (ASV) integrases, and the isolated catalytic core fragment of the ASV protein (residues 52-207). The compound also inhibits retroviral reverse transcriptase in vitro, but the IC(50) for the HIV-1 enzyme is almost two orders of magnitude higher than for HIV-1 integrase. The inhibitor was found to be active in cell culture, preventing reporter gene transduction of HeLa cells by both ASV and HIV-1 vectors. Neither viral attachment nor uptake into cells appeared to be affected in these transfections, whereas accumulation of vector DNA and its joining to host DNA were both drastically reduced in the presence of the inhibitor. Propagation of two different strains of replication-competent HIV-1 in human peripheral blood mononuclear cells (PBMCs) was also reduced by the inhibitor, allowing survival of a substantial number of cells in the treated cultures. Based on these and other results we speculate that binding of 2BrNSA to integrase in infected cells interferes not only with its catalytic activity but also with critical interactions that are required for the formation or function of the reverse transcriptase complex. Its activity in cell culture suggests that this inhibitor may provide a valuable new lead for further development of drugs that target early steps in the HIV life cycle.

Interaction of Staphylococcus epidermidis with endothelial cells in vitro.

Staphylococcus epidermidis is a leading cause of nosocomial bacteremia, yet virtually nothing is known about how this pathogen interacts with human endothelial cells. We present evidence here that two biofilm-producing strains of S. epidermidis adhere to two types of endothelial cell lines in vitro and that adherence is significantly increased after briefly heat-treating the bacteria at 40 degrees C in the presence of calcium. This mild heat treatment resulted in bacteria that were 5 to more than 20 times more adherent than untreated controls. While the adherence of bacteria in all phases of growth was increased after heat treatment, heat-treated late stationary phase cells were generally the most adherent. Electron microscopy demonstrated that S. epidermidis was internalized and appeared to exist free in the cytoplasm. Adherence to endothelium, should it occur in vivo during bacteremia, may be a virulence factor associated with this bacterium's pathogenesis.

Characterization of a monoclonal antibody that binds to an epitope on soluble bacterial peptidoglycan fragments.

We employed an inhibition-type enzyme-linked immunosorbent assay (ELISA) to characterize a murine immunoglobulin M monoclonal antibody (MAb) that bound soluble macromolecular peptidoglycan (PG). With this ELISA, the MAb was capable of detecting soluble PG concentrations of less than 10 ng/ml. Enzymatic digestion of PG reduced binding by more than 100-fold, implying that the epitope recognized by this antibody depended on repeating subunits within the glycan backbone. Additionally, the MAb bound to epitopes on both O-acetylated and non-O-acetylated PG fragments from gram-negative bacteria, as well as PG fragments from Staphylococcus aureus and PG fragments released into the medium by a number of gram-positive and gram-negative bacteria.

Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses.

Retroviral infection induces integrase-dependent apoptosis in DNA-PK-deficient murine scid lymphocytes. Furthermore, the efficiency of stable transduction of reporter genes is reduced in adherent cell lines that are deficient in cellular DNA-repair proteins known to mediate nonhomologous end joining (NHEJ), such as DNA-PK and XRCC4 (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Here we report that wortmannin, an irreversible inhibitor of phosphatidylinositol 3-kinase (PI-3K)-related PKs, including the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) and ATM, sensitizes normal murine lymphocytes to retrovirus-mediated cell killing. We also show that the efficiency of stable transduction of reporter genes in human (HeLa) cells, mediated by either an avian sarcoma virus or a human immune deficiency virus type 1 vector, is reduced in the presence of wortmannin. The dose dependence of such reduction correlates with that for inhibition of PI-3K-related protein kinase activity in these cells. Results from wortmannin treatment of a panel of cell lines confirms that formation and/or survival of transductants is dependent on components of the NHEJ pathway. However, stable transduction is virtually abolished by wortmannin treatment of cells that lack ATM. These results suggest that ATM activity is required for the residual transduction observed in the NHEJ-deficient cells. Our studies support the hypothesis that DNA repair proteins of the NHEJ pathway and, in their absence, ATM are required to avoid integrase-mediated killing [corrected] and allow stable retroviral DNA transduction. The studies also suggest that cells can be sensitized to such killing and stable retroviral DNA integration blocked by drugs that inhibit cellular DNA repair pathways.

The effects of Cryptococcus neoformans-secreted antigens on tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression on human lung epithelial cells.

Since primary infection with Cryptococcus neoformans usually occurs in the lungs, and since pulmonary cryptococcosis involves interactions between yeasts and alveolar epithelial cells, we have begun to study the effects of C. neoformans and its secreted antigens (SA) on epithelial reactions potentially associated with localized inflammation. We report here that SAs from encapsulated and acapsular strains of C. neoformans caused significant reductions in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression on A549 lung epithelial cells in culture. We also present evidence that the reduction in ICAM-1 expression was not associated with SA-induced shedding of this adhesion molecule.

Tourniquet-induced changes of energy metabolism in human skeletal muscle monitored by microdialysis.

BACKGROUND: Tourniquets are often used as part of orthopedic surgery but may cause local and remote organ injury. The authors hypothesized that the procedures used to induce ischemia (circulatory occlusion or exsanguination) may have differential effects on the metabolic state of the muscle that should be reflected in the interstitial levels of metabolites. METHODS: Microdialysis probes were implanted in both quadriceps femoris muscles of 18 patients. Interstitial fluid was obtained during tourniquet-induced ischemia and reperfusion and was analyzed for glucose, lactate, choline, and purines by high-performance liquid chromatography. RESULTS: At a flow rate of 2 microl/min, the average baseline concentrations in the dialysate were 2.5 mM for glucose, 1.7 mM for lactate, 5.2 microM for choline, and 14.3 microM for hypoxanthine. Circulatory occlusion by tourniquet caused a 40% decrease of the extracellular glucose concentration within 30 min. Concomitantly, the interstitial levels of lactate and hypoxanthine increased in a linear fashion to 206% (lactate) and 241% (hypoxanthine) of basal values. The extracellular concentration of choline was also significantly elevated. After exsanguination, the glucose levels were significantly more reduced (by 65%), and the levels of lactate (to 268%) and hypoxanthine (to 286%) were more increased than after circulatory occlusion alone. CONCLUSION: Our microdialysis results demonstrate that the interstitial concentrations of glucose, lactate, and hypoxanthine, which are indicators of tissue ischemia, change more prominently after exsanguination than after circulatory occlusion alone.

Characterization of the self association of Avian sarcoma virus integrase by analytical ultracentrifugation.

Retroviral integration protein (IN) has been shown to be both necessary and sufficient for the integration of reverse-transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-assembly of IN is essential for proper function. Analytical ultracentrifugation was used to determine the stoichiometry and free energy of self-association of a full-length IN in various solvents at 23.3 degrees C. Below 8% glycerol, an association stoichiometry of monomer-dimer-tetramer is observed. At salt concentrations above 500 mM, dimer is the dominant species over a wide range of protein concentrations. However, as physiological salt concentrations are approached, tetramer formation is favored. The addition of glycerol to 500 mM NaCl, 20 mM Tris (pH 8.4), 2 mM beta-mercaptoethanol significantly enhances dimer formation with little effect on tetramer formation. Furthermore, as electrostatic shielding is increased by increasing the ionic strength or decreasing the cation size, dimer formation is strengthened while tetramer formation is weakened. Taken together, the data support a model in which dimer formation includes favorable buried surface interactions which are opposed by charge-charge repulsion, while favorable electrostatic interactions contribute significantly to tetramer formation.

Atomic resolution structures of the core domain of avian sarcoma virus integrase and its D64N mutant.

Six crystal structures of the core domain of integrase (IN) from avian sarcoma virus (ASV) and its active-site derivative containing an Asp64 --> Asn substitution have been solved at atomic resolution ranging 1.02-1.42 A. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144-154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN.

An opsonizing monoclonal antibody that recognizes a noncapsular epitope expressed on Cryptococcus neoformans.

A mouse hybridoma secreting a monoclonal antibody (MAb) that bound a noncapsular epitope expressed on C. neoformans was developed by immunizing BALB/c mice with formalin-killed serotype A yeasts. The hybridoma, designated CSFi, secreted an immunoglobulin G2b MAb that reacted with all C. neoformans serotypes tested, including the acapsular mutant ATCC 52817 (Cap67). Postsectioned immune electron microscopy revealed extensive binding of the MAb to the cell walls of both encapsulated and acapsular yeasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of secreted antigens recovered from concentrated culture supernatants from both encapsulated and acapsular strains was conducted. The results showed that this MAb bound predominantly to antigens with molecular masses of approximately 75 and 100 kDa. A competitive enzyme-linked immunosorbent assay was used to demonstrate that the MAb was not cross-reactive with purified glucuronoxylomannan derived from either serotypes A or D. Experiments conducted with mouse peritoneal phagocytes and the mouse phagocyte-like cell line, J774A.1, demonstrated that the CSFi MAb opsonized the yeasts and increased their adherence to both types of phagocytic cells. We conclude, therefore, that antibodies directed at noncapsular epitopes can serve as opsonins and may have a role in modulating cryptococcal infection.

Indoor air pollution by lindane and DDT indicated by head hair samples of children.

The use of the pesticides DDT and lindane as wood preservatives causes indoor air contamination and subsequently exposure of the occupants of these rooms. As hair acts as a passive sampler for air contaminants, we tested pesticide concentrations in hair measured by the GC/MS technique. A comparison of the DDT and lindane concentrations in hair samples of 193 pre-school children from a rural area around Rostock with information concerning their home environments showed that this is a suitable method for screening purposes.

[Horizontal deceleration trauma with diffuse decollement bleeding--a casuistry]. / Horizontales Dezelerationstrauma mit diffuser Decollementeinblutung--eine Kasuistik.

Treatment of severe haemorrhage caused by multiple trauma is a serious challenge to preclinical as well as clinical management. This is a case report of a motorcycle accident in which a patient sustained total amputation of both legs. Following adequate preclinical care, vital indication led to the patient's immediate surgical treatment. After initially successful haemodynamic stabilisation, the patient developed a horizontal deceleration trauma which resulted in an extended decollement of the muscles of the back and buttock. During the further clinical course, soft tissue bleeding occurred that affected the whole torso. Due to its extent, the bleeding could not be treated surgically, nor did it allow of haemodynamic stabilisation despite continuous massive transfusion. Retrospectively, the impressing amputation injury was treated successfully. In spite of all available surgical and intensive care efforts, however, the slowly demasking monstrous decollement with diffuse tissue bleeding proved to be an injury pattern leading to the patient's death.

Structural basis for inactivating mutations and pH-dependent activity of avian sarcoma virus integrase.

Crystallographic studies of the catalytic core domain of avian sarcoma virus integrase (ASV IN) have provided the most detailed picture so far of the active site of this enzyme, which belongs to an important class of targets for designing drugs against AIDS. Recently, crystals of an inactive D64N mutant were obtained under conditions identical to those used for the native enzyme. Data were collected at different pH values and in the presence of divalent cations. Data were also collected at low pH for the crystals of the native ASV IN core domain. In the structures of native ASV IN at pH 6.0 and below, as well as in all structures of the D64N mutants, the side chain of the active site residue Asx-64 (Asx denotes Asn or Asp) is rotated by approximately 150 degrees around the Calpha---Cbeta bond, compared with the structures at higher pH. In the new structures, this residue makes hydrogen bonds with the amide group of Asn-160, and thus, the usual metal-binding site, consisting of Asp-64, Asp-121, and Glu-157, is disrupted. Surprisingly, however, a single Zn2+ can still bind to Asp-121 in the mutant, without restoration of the activity of the enzyme. These structures have elucidated an unexpected mechanism of inactivation of the enzyme by lowering the pH or by mutation, in which a protonated side chain of Asx-64 changes its orientation and interaction partner.

Structure of the catalytic domain of avian sarcoma virus integrase with a bound HIV-1 integrase-targeted inhibitor.

The x-ray structures of an inhibitor complex of the catalytic core domain of avian sarcoma virus integrase (ASV IN) were solved at 1.9- to 2.0-A resolution at two pH values, with and without Mn2+ cations. This inhibitor (Y-3), originally identified in a screen for inhibitors of the catalytic activity of HIV type 1 integrase (HIV-1 IN), was found in the present study to be active against ASV IN as well as HIV-1 IN. The Y-3 molecule is located in close proximity to the enzyme active site, interacts with the flexible loop, alters loop conformation, and affects the conformations of active site residues. As crystallized, a Y-3 molecule stacks against its symmetry-related mate. Preincubation of IN with metal cations does not prevent inhibition, and Y-3 binding does not prevent binding of divalent cations to IN. Three compounds chemically related to Y-3 also were investigated, but no binding was observed in the crystals. Our results identify the structural elements of the inhibitor that likely determine its binding properties.

Purification of untagged retroviral integrases by immobilized metal ion affinity chromatography.

We have developed a simple protocol for the purification of untagged retroviral integrases expressed in bacterial cells. The method takes advantage of the inherent ability of the proteins to bind metal ions. The protocol involves an initial enrichment of the protein in the pellet fraction following centrifugation of the lysate after cell lysis. Integrase is then solubilized from the pellet at high salt conditions (1 M) with detergent and applied to a nickel-charged iminodiacetic acid-Sepharose column. The enzyme is eluted from the column with imidazole. The resulting protein, which is 70-80% homogeneous, is subsequently purified to homogeneity on a heparin-Sepharose column. The two-column protocol is easily completed in a day and yields approximately 2 mg of enzymatically active protein per gram of wet cell paste.

Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity.

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.

The in vitro interaction of Cryptococcus neoformans with human lung epithelial cells.

The interaction of Cryptococcus neoformans with a human lung epithelial cell line (A549) is described. Encapsulated and acapsular strains adhered to epithelial cells in a time-dependent manner, with the acapsular strain being the most adherent under all conditions tested. Internalized cryptococci were additionally observed. The expression of the adhesins responsible for adherence to the epithelial cells was induced by growth at 37 degrees C. Adhesin expression was repressed in all strains by growth with sucrose as the sole carbon source. A strain-specific repression of adhesin expression was observed after growth with galactose and xylose. A variety of carbohydrates included in the assay suspensions blocked adherence, implicating certain carbohydrate moieties that might serve as ligands for the yeast adhesin. Finally, a monoclonal antibody is described that inhibited cryptococcal adherence to the epithelial cells. Collectively, the results demonstrate a specific interaction between C. neoformans and lung epithelial cells mediated by yeast adhesins whose expression is regulated by environmental factors.