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Biomedical subjects

G Merkel

Publications and source records attributed to G Merkel.

At least 19 recordsLinked to original sources
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↗
PubMed1995

Activities and substrate specificity of the evolutionarily conserved central domain of retroviral integrase.

The retroviral integrase (IN) is a virus-encoded enzyme that is essential for insertion of viral DNA into the host chromosome. In order to map and define the properties of a minimal functional domain for this unique viral enzyme, a series of N- and C-terminal deletions of both Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) INs were constructed. The RSV IN deletion mutants were first tested for their ability to remove two nucleotides from the end of a substrate representing the terminus of viral DNA in order to assess the contribution of N and C regions towards this reaction, referred to as processing. The results suggest that C-terminal amino acids of the intact RSV protein are required to maintain specificity of the processing reaction. Though deficient for processing, the RSV deletion mutants exhibited a secondary endonucleolytic activity that was indistinguishable from that of wild-type IN, demonstrating that all retained some enzymatic activity. RSV, and a larger set of HIV-1, IN deletion mutants were then tested for their ability to perform an intramolecular, concerted cleavage-ligation reaction using an oligodeoxynucleotide substrate that mimics the intermediate viral-host DNA junction found prior to the final step of covalent closure. The composite results from such analyses define a minimal functional central region of approximately 140 amino acids for each enzyme that includes the highly conserved D,D(35)E domain. Results with HIV-1 and HIV-2 IN also indicate that the efficiency of concerted cleavage-ligation depends upon the presence of CA/GT base pairs within the viral component of the DNA substrate at the reaction site. Even the isolated central region of HIV-1 IN exhibited this sequence requirement for optimal activity. We conclude that this evolutionarily conserved central region of IN not only encodes residues that are required for the catalytic activity of the enzyme but also harbors some or all of the determinants responsible for recognition of the CA/GT dinucleotides that are present at the ends of all retroviral DNAs.

Avian Sarcoma Viruses↗
PubMed1995

[Risk for the Rostock water supply from the Warnov river by an old deposit at Schwann?].

Accidents of the city gasworks in the end of the fifties at Schwaan, a small town south of Rostock, resulted in an emission of tar and waste water into the river Warnow, from which the city of Rostock is supplied with drinking water about 20 km downstream. The tar contaminated river sediment (old deposit) covering an area of about 200 m2 has a high pollutant inventory: Steam-distillable phenolic compounds 407 mg/kg in the fresh sediment, polycyclic aromatic hydrocarbons (PAH) 400 to 1100 micrograms/l in the aqueous eluate. The mutagenic potential was detected by the AMES-test. As shown by PAH determinations there were no other pollutant sources in the catchment area, in the tributaries or on other places of the river. The mean PAH concentrations of the river Warnow and his tributaries during 1991 to 1994 were found to be in the range lower than 10 ng/l. Further investigations showed a continuous emission of PAH from the tar laugh in small amounts. An additional emission of pollutants (PAH) and their transportation in the river is expected in the case of sediment disturbances. This emission and their possible consequences are difficult to estimate, even though the seston and the humic substances content of the river seems to reduce the PAH risk. Concluding from our results the probability is very low that pollutants were transported downstream to Rostock and pass the water works but may not totally be excluded. Nevertheless from the viewpoint of care a removal of the old deposit is necessary.

Fresh Water↗
PubMed1994

Indoor factors and IgE levels in children.

The objective of the study was to determine indoor characteristics of households in relation to total serum IgE. In a population-based cross-sectional study, 1096 6-12-year-old children were examined in three East German towns (Eisleben, Hettstedt, and Zerbst). Of the questionnaires, 772 (70.4%) were returned by the parents. Serum IgE of 703 children and urinary cotinine in a random subsample of 224 children were analyzed. Linear regression on log(IgE) adjusted for the main covariates was used to assess indoor risk factors such as room size, and the presence of curtains, carpet, and plants in the child's room. Open-heating facilities indoors, passive smoking, and furniture made of chipboard had the most important effect. A higher urinary cotinine/creatinine ratio was associated with higher total IgE level. Total IGE increased also with the number of persons living in the household, independently of indoor smoking. We conclude that indoor air pollution from smoking and open-heating facilities may increase the IgE levels of children. The role of other factors such as chipboard, which could reflect the emission of formaldehyde, or the number of persons per household, which could reflect viral or helminthic infection, remains to be analyzed.

Air Pollution, Indoor↗
PubMed1993

Histamine release from basophils in childhood: age dependency and inhibition by pertussis infection and pertussis toxin.

The influence of childhood pertussis infection and of purified pertussis toxin on histamine release from human basophil leucocytes was investigated. Three different stimuli, the peptide N-formyl-Met-Phe (NFMP), anti-IgE, and the calciumionophore A23187 were used to challenge the cells. When NFMP was the stimulus, histamine release in the control group (age 0.5-17 years) increased in an age-dependent fashion, whereas anti-IgE and A23187 stimulated release did not vary with age. During the convulsive state of pertussis infection there was a significant reduction of histamine release in response to 10 microM NFMP (from 9.5 +/- 1.4 [n = 21] to 6.7 +/- 1.5 [n = 19], P < 0.05) and in response to 800 and 80 U/ml anti-IgE (from 28.5 +/- 5 [n = 19] to 16.3 +/- 5 [n = 13], P < 0.05, and from 6.9 +/- 1.7 [n = 16] to 2 +/- 0.8 [n = 13], P < 0.01), whereas histamine release stimulated by A23187 was unchanged compared to release in control children. In vitro pretreatment of basophils from healthy children and adults with pertussis toxin also inhibited histamine release. When NFMP was the stimulus, release was completely blocked by pertussis toxin with an IC50 of about 11 ng/ml, whereas anti-IgE stimulated release was only inhibited by 20%-30% and release induced by A23187 was reduced to 40%-50% by toxin treatment. In conclusion we have demonstrated a functional impairment of histamine release during the convulsive state of pertussis and that this inhibition is likely to be mediated by pertussis toxin.

Adolescent↗
PubMed1993

[Isolated proximal fibula fracture in Marfan syndrome].

An isolated fracture of the proximal fibular shaft without associated tibial or ankle injury and without resulting from direct violence is extremely rare. This report presents a case of an unusual isolated proximal fibular fracture (stress-fracture) in a female tennis-player with Marfan-syndrome. The literature is reviewed and diagnosis, treatment and etiology are discussed.

Adult↗