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J Fetzer

Publications and source records attributed to J Fetzer.

14 recordsLinked to original sources
PubMed2000

A bidomain model based BEM-FEM coupling formulation for anisotropic cardiac tissue.

A hybrid boundary element method (BEM)/finite element method (FEM) approach is proposed in order to properly consider the anisotropic properties of the cardiac muscle in the magneto- and electrocardiographic forward problem. Within the anisotropic myocardium a bidomain model based FEM formulation is applied. In the surrounding isotropic volume conductor the BEM is adopted. Coupling is enabled by requesting continuity of the electric potential and the normal of the current density across the boundary of the heart. Here, the BEM part is coupled as an equivalent finite element to the finite element stiffness matrix, thus preserving in part its sparse property. First, continuous convergence of the coupling scheme is shown for a spherical model comparing the computed results to an analytic reference solution. Then, the method is extended to the depolarization phase in a fibrous model of a dog ventricle. A precomputed activation sequence obtained using a fine mesh of the heart was downsampled and used to calculate body surface potentials and extracorporal magnetic fields considering the anisotropic bidomain conductivities. Results are compared to those obtained by neglecting in part or totally (oblique or uniform dipole layer model) anisotropic properties. The relatively large errors computed indicate that the cardiac muscle is one of the major torso inhomogeneities.

Animals↗
PubMed1997

Oligonucleotide-directed mutagenesis and subsequent expression of the corresponding recombinant proteins without changing the bacterial vector system.

A new bacterial vector was constructed that combines the attractive features of gene fusion vectors and phagemids. A gene of interest cloned into this new vector can either be expressed as fusion protein or be prepared as single-stranded template DNA within the same system. Thus, time consuming subcloning procedures changing the bacterial vector according to the required method are avoided. As an example sequencing, expression and subsequent purification of site-directed mutants of herpes simplex virus type 1 thymidine kinase are discussed.

Cloning, Molecular↗
PubMed1995

Site-directed mutagenesis of herpes simplex virus type 1 thymidine kinase opposes the importance of amino acid positions 251, 321 and 348 for selective recognition of substrate analogs.

Seven site-directed mutants representing step-by-step transitions from the thymidine kinase (TK) of Herpes Simplex Virus type 1 (HSV 1) strain F to that one of strain SC16 were constructed, recombinantly produced and kinetically characterized in order to identify which of three differences in the amino acid sequence of these two TKs is/are responsible for their difference in substrate specificity. The preference of these two TKs for the substrate analogs aciclovir and ganciclovir was reported to be in reverse order (4.5), suggesting one of the amino acids in position 251 (cys or gly), 321 (ser or pro) and 348 (val or ile) of the HSV 1 wildtype TKs to be important for selective substrate recognition. However, the results of our study do not support this hypothesis.

Acyclovir↗
PubMed1994

Site-directed mutagenesis clarifies the substrate position within the three-dimensional model of the active site of herpes simplex virus type-1 thymidine kinase.

Site-directed mutagenesis was used to experimentally verify the 3D model of the active site of herpes simplex virus type-1 thymidine kinase (HSV 1 TK) obtained by homology modelling. For this purpose, D215 and K317 were replaced by R and G, respectively, at homologous positions in the aciclovir-insensitive bovine herpes virus type-1 thymidine kinase (BHV 1 TK). Wild-type and mutated enzymes were expressed in Escherichia coli using a gene fusion vector and purified to homogeneity. While both mutants had the same Km value for thymidine as the recombinant wild-type enzyme (0.2 microM), Vmax was decreased to 20-25% of the original wild-type value. The recombinant wild-type enzyme was inhibited by the substrate analogue aciclovir with a Ki of 146 microM. Both mutants were able to phosphorylate aciclovir to about the same extent as the wild-type enzyme. These findings suggest that neither D215 nor K317 are directly involved in substrate binding. Therefore, a rearrangement of the 3D model is suggested, concerning the assignment of the substrate-binding site and co-substrate-binding site at the right and left side of the phosphate-binding loop, respectively.

Base Sequence↗
PubMed1994

A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase.

Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein. The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography. Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing. In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine. Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers. Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.

Amino Acid Sequence↗
PubMed1993

Site-directed mutagenesis in the active site of the herpes simplex virus type 1 thymidine kinase gene.

The thymidine kinase (TK) of herpes simplex virus type 1 (HSV-1) contains three regions of homology to other ATP utilizing enzymes. We have altered one region of the protein, which seems to play an important role in phosphorylation substrates by site-directed mutagenesis. When the aspartate 162 was changed to asparagine, the enzyme lost its activity. To identify the inactive protein, expressed by a vaccinia vector in eukaryotic cells, a monospecific antiserum against a bacterial tryptophan E-HSV-1 TK fusion protein was made. These results support the suggestion that aspartate 162 is essential for the enzymatic activity.

Amino Acid Sequence↗
PubMed1991

Computer-aided active-site-directed modeling of the herpes simplex virus 1 and human thymidine kinase.

Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core beta-sheet structure, connected by loops and alpha-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding site is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site into Asn. First investigations reveal that the enzymatic activity of the mutant protein is destroyed.

Amino Acid Sequence↗