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Biomedical subjects

G Merkel

Publications and source records attributed to G Merkel.

At least 19 recordsLinked to original sources
PubMed2006

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.

Adult↗
PubMed2005

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 (<or=2.40), urine collected on Monday were statistically significant associated with high nicotine and cotinine excretion levels. Children exposed to parental smoke showed much higher biomarker levels than the non-smoking spouse of an adult smoker. Therefore, children need specifically protection from ETS at home. PRACTICAL IMPLICATIONS: As children showed a higher internal exposure compared with adults the health hazards association with passive smoking might have a more serious effect in children. Children are unable to complain and unable to remove themselves from tobacco smoke exposure. Thus, parents should make sure that their children live in a smoke free environment. Each country should take all necessary legislative and regulatory measures which forbids smoking in public places and the home environment even if it requires sacrifices by the parents.

Adolescent↗
PubMed2004

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.

Avian Sarcoma Viruses↗
PubMed2001

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.

Androstadienes↗
PubMed2000

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.

Adult↗
PubMed1999

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.

Avian Sarcoma Viruses↗
PubMed1999

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.

Amino Acid Substitution↗
PubMed1999

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.

Air Pollution, Indoor↗
PubMed1999

[Horizontal deceleration trauma with diffuse decollement bleeding--a casuistry].

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.

Accidents, Traffic↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

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.

Avian Sarcoma Viruses↗
PubMed1998

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.

Avian Sarcoma Viruses↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1996

Targeting of retroviral integrase by fusion to a heterologous DNA binding domain: in vitro activities and incorporation of a fusion protein into viral particles.

Retroviral DNA integration is catalyzed by a viral protein, the integrase (IN). IN recognizes sequences at the viral DNA ends, specifically nicks these ends (the processing reaction), and inserts them into host DNA (the joining reaction). The mechanism by which host DNA integration sites are selected is unknown, although it is clear that many regions are accessible to the retroviral integration machinery. To investigate integration site selection, and to possibly influence this process, we have used a model system in which the avian sarcoma virus (ASV) IN, and segments thereof, have been fused to the Escherichia coli LexA repressor protein DNA binding domain (DBD). We provide evidence that a variety of such IN-LexA DBD fusion proteins can be directed to cognate lexA operators in vitro. Binding of precise N- and C-terminal fusion proteins to the operators is associated with a significant increase in the joining of viral DNA to sites immediately adjacent to the lexA operator region. We also carried out domain exchange and deletion experiments which revealed that the highly conserved "zinc-finger" domain of ASV IN is not essential for processing of viral DNA ends or joining of viral DNA ends to DNA target sequences. Last, the C-terminal fusion was engineered into the viral genome and we show that this protein can be incorporated into viral particles. Our results suggest that it might be possible to augment IN function in vivo through a heterologous domain. These observations have implications for retroviral-mediated gene therapy.

Amino Acid Sequence↗
PubMed1996

The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations.

BACKGROUND: Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity. So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E. coli and human immunodeficiency virus 1 (HIV-1) RNase H. Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes. RESULTS: The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution. A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination. The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN. CONCLUSIONS: Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN. This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site. Only a single divalent cation was observed even at extremely high concentrations of the metals. We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism.

Avian Sarcoma Viruses↗
PubMed1996

Hazards for the Rostock water supply from a tar-contaminated sediment in the river Warnow.

By the gas plant at Schwaan (a small city near Rostock), tar and other by-products were released into the river Warnow which supplies Rostock with drinking water 20 km downstream. The tar-contaminated river sediment contained phenols and polycyclic aromatic hydrocarbons (PAH), indicating a mutagenic potential. Investigations in the catchment area and on tributaries showed PAH contents up to about 10 ng/l in the water. The river sediment was found to be the sole PAH source. Although the PAH level is very low, sediment disturbances may produce an increase in concentrations. The consequences of such a worst case are difficult to estimate, even though the content of seston and humic substances in the river seems to reduce the PAH concentrations. Although this lowers the probability that increased PAH concentrations reach the Rostock water supply, the removal of the tar-contaminated sediment from the river is strongly recommended.

Environmental Monitoring↗
PubMed1995

High-resolution structure of the catalytic domain of avian sarcoma virus integrase.

Retroviral integrase (IN) functions to insert retroviral DNA into the host cell chromosome in a highly coordinated manner. IN catalyzes two biochemically separable reactions: processing of the viral DNA ends and joining of these ends to the host DNA. Previous studies suggested that these two reactions are chemically similar and are carried out by a single active site that is characterized by a highly conserved constellation of carboxylate residues, the D,D(35)E motif. We report here the crystal structure of the isolated catalytic domain of avian sarcoma virus (ASV) IN, solved using multiwavelength anomalous diffraction data for a selenomethionine derivative and refined at 1.7 A resolution. The protein is a crystallographic dimer with each monomer featuring a five-stranded mixed beta-sheet region surrounded by five alpha-helices. Based on the general fold and the arrangement of catalytic carboxylate residues, it is apparent that ASV IN is a member of a superfamily of proteins that also includes two types of nucleases, RuvC and RNase H. The general fold and the dimer interface are similar to those of the analogous domain of HIV-1 IN, whose crystal structure has been determined at 2.5 A resolution. However, the ASV IN structure is more complete in that all three critical carboxylic acids, Asp64, Asp121 and Glu157, are ordered. The ordered active site and the considerably higher resolution of the present structure are all important to an understanding of the mechanism of retroviral DNA integration, as well as for designing antiviral agents that may be effective against HIV.

Amino Acid Sequence↗