Biomedical subjects

B Schwenzer

Publications and source records attributed to B Schwenzer.

9 recordsLinked to original sources

PubMed2007

Surface modification of titanium-based alloys with bioactive molecules using electrochemically fixed nucleic acids.

A new method of surface modification for titanium (alloys) with bioactive molecules was developed with the intention of providing a new basis of implant adaptation for particular requirements of certain medical indications. Nucleic acid single strands are fixed electrochemically via their termini (regiospecifically) by growing an oxide layer on Ti6Al7Nb anodically. It could be shown that they are accessible to subsequent hybridization with complementary strands at physiological pH. Amount of nucleic acids immobilized and hybridized were determined radioanalytically using 32P-labelled nucleic acids. Stable fixation was attained at and above potentials of 4 V(SCE). Up to 4 pmol/cm2 of nucleic acid single strands could be immobilized and hybridization efficiencies up to 1.0 were reached. Hybridization efficiency was found to depend on surface density of immobilized oligonucleotides, while hybridization rates increased when MgCl2 was added. A conjugate consisting of an oligonucleotide complementary to the immobilized strand and the hexapeptide GRGDSP with RGD as an integrin recognition site was synthesized. This conjugate was able to bind to integrins on osteoblasts. It was shown that this conjugate binds to the anchor strand fixed on Ti6Al7Nb to an extent comparable with the unconjugated complementary strand.

Animals↗

PubMed1999

Oligonucleotide facilitators enhance the catalytic activity of RNA-cleaving DNA enzymes.

From in vitro selection studies, DNA structures have been found that cleave target RNA sequence specifically and show a certain similarity to the well-investigated hammerhead ribozymes. Such DNA enzymes are more resistant to nuclease-mediated degradation than RNA enzymes. On the other hand, their cleavage activity is lower than the activity of hammerhead ribozymes. In the present study, we improved the activity of DNA enzymes by adding oligonucleotide facilitators complementary to the 5' and the 3' ends of the substrate to the cleavage reaction. DNA enzyme activity in vitro was monitored under multiple turnover conditions using short RNA model substrates. We have shown that oligonucleotide facilitators strongly enhance the multiple turnover activity of the DNA enzyme reaction. In one of our model systems with a suitable facilitator combination, we were able to observe a more than 200-fold enhancement of the k(cat)/Km value. The comparison of two DNA enzyme-substrate systems showed that the principal effects of the facilitators were independent of the substrate sequence. However, the degree of facilitator effect was noticeably dependent on the basic catalytic efficiency of DNA enzymes. Furthermore, the efficiency of the DNA enzyme reaction with facilitator was compared with the reaction of a DNA enzyme with a stem sequence extended by the sequence of the facilitator. The multiple turnover activity of such a "long DNA enzyme" is higher than the activity of the short DNA enzyme without facilitators. However, when compared with the multiple turnover reactions of the short DNA enzyme with facilitator, the reaction with the long DNA enzyme is considerably slower. The results obtained with our model systems demonstrate that oligonucleotide facilitators enable DNA enzymes to act as effective multiple turnover catalysts by cleavage of RNA substrates.

Base Sequence↗

PubMed1998

Oligonucleotide facilitators enable a hammerhead ribozyme to cleave long RNA substrates with multiple-turnover activity.

Trans-acting hammerhead ribozymes are usually efficient in cleaving short RNA model substrates under both single-turnover and multiple-turnover conditions. In contrast, when long RNAs are the substrates, the cleavage efficiency of these ribozymes decreases, including a loss of multiple-turnover activity in many cases. Since target substrates for potential therapeutical purposes are mostly long RNAs, a multiple-turnover cleavage of long RNAs would essentially increase the efficiency of hammerhead ribozymes. Therefore, we explored if oligonucleotide facilitators, capable of enhancing multiple-turnover activity with short substrates, can also affect or cause multiple turnover with long substrates. We examined the effects of 12-base and 24-base oligonucleotide facilitators on the multiple-turnover activity with substrates of different length containing 39-, 452- and 942-base sequences of the human tissue factor (HTF) mRNA. In the absence of facilitator, the ribozyme cleaved only the 39-base substrate with multiple-turnover activity, but not the long 452-base and 942-base substrates. However, facilitator addition enabled the ribozyme to cleave even the 452-base and the 942-base substrates with multiple-turnover activity. All facilitators tested showed a remarkable activating effect with the long substrates. The data demonstrate that a hammerhead ribozyme which, by itself, can only act as a single-turnover catalyst with long substrates, can be switched by facilitators into a multiple-turnover catalyst. Thus, the inactivation of long target RNAs in multiple-turnover reactions may be achieved by addition of oligonucleotide facilitators.

Humans↗

PubMed1997

Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes.

Humans↗

PubMed1996

Efficient improvement of hammerhead ribozyme mediated cleavage of long substrates by oligonucleotide facilitators.

Hammerhead ribozymes were found to be not very efficient in cleaving long RNA substrates in trans. Oligonucleotide facilitators, capable of affecting hammerhead ribozymes by interacting with the substrate at the termini of the ribozyme, may improve this reaction. We determined in vitro the effects of 18 DNA and RNA oligonucleotide facilitators on three substrates containing 39, 452, and 942 nucleotides, respectively, by estimating the facilitator influences on association between ribozyme and substrate and on the cleavage step. The effects increase with the length of the substrates. With the 39mer substrate a maximal 4-fold enhancement of the ribozyme activity could be detected, the reaction with the 942mer substrate was accelerated up to 115-fold by facilitator addition. In long, structured substrates the facilitators have the potential to preform the substrate for the ribozyme attack. Due to this preforming effect, the rate of ribozyme-substrate association was increased as well as the rate of the cleavage step. 3'-End facilitators accelerate both of these rates, largely independent on the facilitator length. The rate of the cleavage step is raised as a result of a favorable activation energy gain by these facilitators. With all substrates, the 5'-end facilitators increase the association rate between ribozyme and substrate in dependence on their length. With the 39mer substrate the 5'-end facilitators decrease the rate of the cleavage step. With the long substrates 5'-end facilitators partially increase the rate of the cleavage step due to their preforming potential with these substrates. In some examples, combinations of several 5'-end and 3'-end facilitators provide an additional improvement over single facilitators in both the association between ribozyme and substrate and the cleavage step. Results suggest that even short facilitators may be efficient effectors enhancing hammerhead ribozyme mediated cleavage of long substrates.

Base Sequence↗

PubMed1996

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems.

Base Sequence↗

PubMed1989

Study on the partition of beta-galactosidase from Escherichia coli in aqueous two-phase systems and elaboration of a simple procedure for the purification of the enzyme to homogeneity.

The partition of beta-galactosidase from Escherichia coli in aqueous two-phase systems composed of poly(ethylene glycol)/Aquaphase PPT or poly(ethylene glycol)/potassium phosphate, respectively, has been studied thoroughly by varying diverse parameters of the phase forming conditions. The enzyme was found to partition in both systems predominantly to the upper poly(ethylene glycol) containing phase, while the protein bulk of E. coli is concentrated in the lower phase. Optimum conditions for the employment of the two-phase partition for the purification of the enzyme were elaborated leading to a simple and effective procedure in which the enzyme was isolated to homogeneity with 70% recovery.

Chromatography, DEAE-Cellulose↗

PubMed1987

Large scale purification of intralumenal alkaline phosphatase from calf intestine by immunosorbent affinity chromatography.

Electrophoretically and immunologically homogeneous alkaline phosphatase (AP) can be obtained by a simple and rapid two-step procedure. Step 1: Immunosorbent affinity chromatography using immobilized polyclonal anti-AP-antibodies and elution of the bound enzyme by triethylamine solution, pH 11.4. After affinity chromatography the specific activity of the extracted crude material increases from 3.5 to 793 IU/mg. Step 2: Final purification to a specific activity of 1524 IU/mg by DEAE-cellulose ion exchange chromatography. One investigator is able to purify 100 mg AP within 3 days. The overall recovery is 65%. Methods for characterization and selection of anti-AP-antiserum using immunoinhibition of AP are described.

Alkaline Phosphatase↗

PubMed1982

Hydrogenolytic debenzylation of sulfur-containing peptides.

S-protected cysteine derivatives are desulfurized by hydrogenation with Pd/H2 forming inhibiting thiols and alanine derivatives. In the case of protected cysteine peptides, this inhibition effect may be suppressed by adding BF3-etherate, the extent of suppression depending on the position of the cysteine and on the sequence length of the peptide derivative. With sufficient sequence distance from the cysteine sulfur, benzyl type protecting groups may be quantitatively removed from the cysteine sulfur by means of hydrogenolysis.

Benzyl Compounds↗