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Biomedical subjects

K H Hecker

Publications and source records attributed to K H Hecker.

8 recordsLinked to original sources
PubMed1999

Distinct roles for transforming growth factor-beta2 and tumour necrosis factor-alpha in immune deviation elicited by hapten-derivatized antigen-presenting cells.

The role of antigen-presenting cells (APC) in the induction of antigen-specific unresponsiveness was examined, using two functionally distinct murine macrophage hybridomas, #59 and #63 cells. Derivatized with the hapten (dinitrofluorobenzene; DNFB), #59 cells induced contact hypersensitivity (CH) in mice. Hapten-derivatized #63 cells failed to induce CH. Instead, they prevented recipients from acquiring CH when exposed subsequently to a sensitizing dose of the hapten. Similarly, hapten-derivatized #59 cells, pretreated in vitro with transforming growth factor-beta2 (TGF-beta2) lost their capacity to evoke CH, and induced tolerance. Hapten-derivatized #63 cells and TGF-beta2-treated #59 cells eliminated CH in mice sensitized to hapten. Reverse transcription-polymerase chain reaction analysis of mRNAs for various accessory molecules important in T-cell activation revealed that #63 and TGF-beta2-treated #59 cells differed only in their expression of tumour necrosis factor-alpha (TNF-alpha) mRNA. The latter expressed higher levels of TNF-alpha mRNA than did untreated #59 cells. As a consequence, #63 and TGF-beta2-treated #59 cells, both of which induce tolerance, secrete TNF-alpha protein unlike untreated #59 cells, which do not induce tolerance to hapten. Since neutralizing anti-TNF-alpha antibodies abrogated the tolerogenic potential of #63 cells in vivo, we conclude that TGF-beta2 equips hapten-bearing APC with the capacity to evoke systemic immune deviation in which CH is selectively silenced. We speculate that one effect of TGF-beta2 is to cause APC to up-regulate TNF-alpha production. In turn, this cytokine biases the functional property of responding hapten-specific T cells in a direction that not only interferes with acquisition, but suppresses induction of CH.

Animals↗
PubMed1998

Error analysis of chemically synthesized polynucleotides.

Two single-stranded polynucleotide constructs, 123 and 126 nucleotides in length, were chemically synthesized using standard phosphoramidite chemistry. Clonable, double-stranded DNA fragments about 100-bp long were prepared from the polynucleotides by primer extension with a DNA polymerase and end-trimming with two restriction endonucleases, then the fragments were ligated into separate plasmids. Errors in individual insert copies were determined by dideoxy sequencing after in vivo amplification of plasmids. Five of the ten inserts sequenced contained errors, including seven single-base-pair deletions, one four-base-pair deletion and one G-->C transversion. The origins of the latter two errors are unclear, but single-base deletions are inconsistent with errors of polymerases; thus, the most common sequence errors of chemical synthesis are deletion mutations. Deletions are most likely to result from incomplete capping or de-tritylation. The observed error rate can became a significant limiting factor in applications that depend on the correctness of a polynucleotide sequence in individual insert clones.

Cloning, Molecular↗
PubMed1997

Synthetic polynucleotide templates for characterizing sequence-selective small molecule/nucleic acid interactions.

A synthetic polynucleotide sequence was developed and cloned to serve as a target in footprinting and other assays designed to characterize the sequence-selective binding of drugs and other small molecules to various forms of nucleic acids. The target sequence comprehensively represents all base quartet recognition sites in a minimal length sequence. Minimal length target sequences were found to be 144 nt long. One such target sequence was divided into two parts. One strand of each part was chemically synthesized and the complementary strands were generated using a DNA polymerase. Double-stranded sequences were then cloned into pGEM-3Zf(+/-) vectors (Promega, Inc.). The cloned target sequence can be used directly in double-stranded DNA form. Alternatively, features of the plasmid vector allow expression of the target sequences as single-stranded DNA or RNA or as RNA/DNA or RNA/RNA duplexes. These cloned target sequences designed for high information content overcome limitations to the use of natural DNA sequences for footprinting and related experiments arising from the unequal representation of base quartets and the potential for secondary structure formation in single-stranded forms.

Base Sequence↗
PubMed1996

Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptase and other polymerases.

Primer extension assays using recombinant templates constructed to contain all 256 possible base quartets in a minimum length sequence were used to examine binding of the anticancer drug actinomycin D to single-stranded DNA. Single-stranded templates were generated by digestion of linearized plasmid with the double-strand-specific T7 gene 6 exonuclease. Actinomycin D formed high-affinity, kinetically stable complexes that paused primer elongation at specific sites by HIV-1 reverse transcriptase, Sequenase (modified T4 DNA polymerase), the Klenow fragment of Escherichia coli DNA polymerase, and Vent (exo-) DNA polymerase. Pauses occurred most commonly near G+C-rich nucleotide clusters, including GpC steps, the preferred sites of double-stranded DNA binding. Complexes were stable for several minutes at temperatures over 50 degrees C as determined by their abilities to pause Vent polymerase at elevated temperatures. Significant variations were noted in pause patterns of different polymerases, demonstrating differential responses of polymerases to a bound actinomycin. Covalent adducts formed on template DNA by a photoaffinity analog of actinomycin D completely stopped primer extension. These results support the possibility that actinomycin D inhibits transcription elongation by complexing single-stranded DNA in the open transcription complex. Single-stranded DNA binding by actinomycin D or analogs may also provide routes for combating HIV or other viruses which replicate through single-stranded intermediates.

Base Composition↗
PubMed1996

Selective DNA binding of (N-alkylamine)-substituted naphthalene imides and diimides to G+C-rich DNA.

Alkylamine-substituted naphthalene imides and diimides bind DNA by intercalation and have applications as anticancer agents. The unique structures of these imides in which two adjacent carbonyl groups lie coplanar to an extended aromatic ring system allow the possibility of sequence-selective interactions between the intercalated chromophore and guanine amino groups situated in the DNA minor groove. The binding affinities of N-[3-(dimethylamino)propyl amine]-1,8-naphthalenedicarboxylic imide (N-DMPrNI) and N,N'-bis [3,3'-(dimethylamino)propylamine]-naphthalene-1,4,5,8-tetracarboxylic diimide (N-BDMPrNDI) for natural DNAs of differing base composition were determined spectroscopically and by equilibrium dialysis. In agreement with the above proposition, binding studies indicated that both the naphthalene imide and diimide strongly prefer to intercalate into steps containing at least one G:C base pair. The dependencies of association constants on DNA base composition are consistent with a requirement for one G:C pair in the binding site of the monomide, and two G:C pairs in binding sites of the diimide. These selectivities are comparable to or exceed that of actinomycin D, a classic G:C-selective drug. Protection footprinting with DNase I confirmed that the naphthalene monoiimide (N-DMPrNI) prefers to bind adjacent to G:C base pairs, with a most consistent preference for "mixed" steps containing both a G:C and an A:T pair, excepting GA:TC. Several 5'-CG-3' steps were also good binding sites as indicated by nuclease protection, but few GC:GC or GG:CC steps were protected. The naphthalene diimide inhibited DNase I digestion, but did not yield a footprint. The base recognition ability and versatile chemistry make naphthalene imides and diimides attractive building blocks for design of highly sequence-specific, DNA-directed drug candidates including conjugated oligonucleotides or oligopeptides.

Amines↗
PubMed1996

High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR.

Touchdown (TD) PCR represents a versatile one-step procedure for optimizing PCRs even if the degree of primer-template complementarity is not fully known. The protocol relies on incremental annealing temperature decreases in progressive cycles designed to bracket the melting temperature (Tm) of the reaction. Here we investigate the characteristics of TD PCR that serve to minimize the need to optimize annealing temperature or buffer conditions and yet produce single strong target amplicons. We demonstrate that priming initiates above the optimum annealing temperature; this helps to ensure a competitive advantage for the target amplicon. On the other hand, as the cycling program progresses, annealing temperatures well below the Tm can serve to significantly increase yields in reactions that would otherwise be marginal due to suboptimal buffer composition and yet do not promote spurious amplification. Modified forms of TD PCR, termed stepdown PCR, consisting of fewer but steeper incremental declines in annealing temperature, are also shown to be effective and can simplify thermal cycler programming.

Animals↗