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Biomedical subjects

G Sczakiel

Publications and source records attributed to G Sczakiel.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mechanistic insights into p53-promoted RNA-RNA annealing.

The tumour suppressor protein p53 promotes the annealing of complementary nucleic acids in vitro. We observed an up to 1600-fold increase of RNA-RNA annealing by recombinant p53 protein which was shown to bind to RNA in sequence-independent way. Nuclease mapping experiments suggest that p53 binds to intramolecular duplex portions and only marginally changes the overall secondary structure of RNA at conditions of increased annealing. Thus, the mechanism of p53-promoted RNA-RNA annealing does not seem to be dependent on an activity that melts or changes RNA structure. The activation enthalpy of RNA-RNA annealing is decreased in the presence of p53, i.e. the p53 protein could stabilize the transition state whereas the activation entropy is unfavourable. A comparison with thermodynamic data measured for other facilitators strongly suggests that the mechanism of p53-promoted RNA-RNA annealing is distinct from the mechanism by which other facilitators work. The annealing activity of p53 is almost abolished in the presence of magnesium indicating that it can be sharply regulated in vitro and, in principle, could also be regulated in vivo.

Escherichia coli↗
PubMed1997

The hepatitis B virus posttranscriptional regulatory element contains a highly stable RNA secondary structure.

Hepatitis B virus (HBV) transcripts contain a sequence known as the posttranscriptional regulatory element (PRE). This element was shown to facilitate the nuclear export of the S gene transcripts, to partially substitute for the human immunodeficiency virus (HIV-1) Rev-response element (RRE), and to bind two cellular factors. Within the genetically defined PRE (approximately 450 nucleotides), we identified a highly stable secondary structural element of 313 nucleotides in length termed PRE313. The energy values of the PRE313 are similar to those of the RRE of HIV-1 and significantly lower than those of other portions of HBV RNA. A comparison of human HBV subtypes shows strong conservation of the PRE313 in terms of energy and structure, providing further evidence for the biological significance of the genetically defined PRE and the PRE313 in particular. The structural model for the PRE313 described in this study may help in identifying crucial components of the transport mechanism of transcripts of HBV.

Base Sequence↗
PubMed1997

The subcellular localization and length of hammerhead ribozymes determine efficacy in human cells.

The length requirements of the antisense portion of hammerhead ribozymes for efficacy in living cells was investigated. The HIV-1tat-directed asymmetric hammerhead ribozyme alphaYRz195 was used with a 195 nt 3'-antisense arm and a 3 nt 5'-antisense portion as well as a set of successively 3'-shortened derivatives thereof. In the 3'-antisense arm a minimum length of 20 complementary nucleotides was required for efficient association with a 645 nt target RNA transcript in vitro(for all constructs kass ranged between 0.3 and 1.8x104/M/s). The cleavage rate constants (kcleav) were independent of the length of the antisense flank and ranged between 0.8 and 1.2x10-4/s. However, the length of the antisense arms, as well as the mode of delivery and the subcellular location of the ribozymes, had a dramatic effect on efficacy in HIV-1-producing human cells. When proviral HIV-1 DNA and ribozymes were co-microinjected into the nucleus of human cells, a minimum length of 51 nt in the antisense arm was necessary for antisense- and ribozyme-mediated inhibition of HIV-1 replication. Ribozymes with shorter antisense arms were almost ineffective. Conversely, short chain ribozymes, including those with chemical modifications, were superior to long chain ribozymes when co-microinjected into the cytoplasm. When transfected, all ribozymes showed an antisense effect as well as an additional ribozyme-mediated increase in inhibition. Consequences for the design and application of ribozymes are discussed.

Base Sequence↗
PubMed1997

Purification and characterization of an active form of the p78Rep protein of adeno-associated virus type 2 expressed in Escherichia coli.

The 78-kDa product (p78Rep) of the rep gene of AAV-2 was expressed with an amino-terminal histidine-tag in Escherichia coli and was purified under denaturing conditions. After renaturation of the p78Rep protein by serial steps of dialysis, the biochemical activities of the p78Rep protein were demonstrated, which include the ATP-dependent endonuclease and helicase activity as well as sequence-specific binding to the AAV-2 terminal repeat. These activities were retained when the protein was purified under denaturing conditions followed by renaturation. When compared with published data for p68Rep, the helicase activity of p78Rep was stronger and the endonuclease activity was weaker. The p78Rep protein was able to inhibit HIV-1 replication after co-microinjection together with infectious proviral HIV-1 DNA into the nuclei of human cells, suggesting that p78Rep is necessary for inhibition of HIV-1 in vivo.

Cell Line↗
PubMed1997

Disinfection of cell-associated and extracellular HIV-1 by PUVA treatment.

To inactivate cell-associated and extracellular HIV-1 while preserving cellular surface antigens, a procedure was used based on PUVA treatment, i.e. addition of psoralen to cell suspensions followed by irradiation with UVA light. T-lymphoid MT-4 cells were infected with HIV-1 strain NL4-3, 4'-aminomethyl-4,5',8-trimethylpsoralen was added, and the cell suspension was irradiated with 20 mW/cm2 UVA light for 3, 4 and 5 min. To evaluate virus inactivation, cells and supernatants were diluted serially and cocultured with uninfected MT-4 cells. Infectious HIV-1 was detected by cytopathic effects, immunofluorescence and p24 antigen ELISA. UVA irradiation at 3.6 J/cm2 (3 min 20 mW/cm2) reduced the amounts of both cell-associated and extracellular infectious HIV-1 by more than five orders of magnitude. Even at more stringent conditions of PUVA treatment (10 min 20 mW/cm2 UVA irradiation), conformational cellular surface epitopes remained detectable by flow cytometry.

Anti-HIV Agents↗
PubMed1997

The design of antisense RNA.

Antisense nucleic acids comprise short-chain synthetic oligonucleotides, often oligodeoxyribonucleotides (ODN) of less than approximately 30 nucleotides and substantially longer sequences formed by ribonucleic acids (RNA). Both groups differ with respect to several properties, including their generation, the mode of delivery, and their structure-function relationship. Long-chain antisense RNA transcribed in vitro or endogenously from recombinant genes fold into three-dimensional structures. The pairing reaction with their complementary target strand occurs via largely unknown annealing mechanisms and, depending on the phylogenetic cellular background, in different cellular compartments. The annealing pathway is influenced by a variety of biologic and biochemical parameters that are as yet poorly understood. However, the basal biochemical mechanisms underlying the relationship between RNA structure and efficient annealing could allow one to derive more general rules for the design of in vivo effective antisense RNA in a way that is not dependent on specific cell types. Here, some of the criteria are discussed that are currently thought to have major impact on the design of long-chain antisense RNA.

Animals↗
PubMed1997

Hammerhead ribozyme activity in the presence of low molecular weight cellular extract.

Hammerhead ribozymes cleave RNA in vitro at magnesium concentrations that are not thought to be available in vivo. To search for cellular factors that could substitute for the role of magnesium, we investigated the influence of size-fractionated cell extracts on the rate of association with its target and the cleavage rate of the HIV-1-directed long-chain hammerhead ribozyme alphaYRz60. When using a fraction containing cellular compounds smaller than a molecular weight cutoff of 5000 (MWCO5000), we observed no influence on the annealing of the ribozyme with its target but strongly increased cleavage activity under single turnover conditions. The cleavage rate constant in the presence of the MWCO5000 extract was similar to the effect of approximately 25 mM magnesium in vitro, indicating that hammerhead ribozyme cleavage could occur at considerable rates in vivo.

Cell Extracts↗
PubMed1997

Theoretical and experimental selection parameters for HBV-directed antisense RNA are related to increased RNA-RNA annealing.

Annealing kinetics of antisense species against two different target regions of the hepatitis B virus (HBV) were measured by kinetic in vitro selection. Individual association rates were related to energies calculated for local sequence segments and predicted structures of the complete pregenomic target RNA. A relationship between the presence of external loops and joint sequences with fast pairing was observed whereas internal loops did not favor fast RNA-RNA annealing. The findings were used to predict a fast-annealing HBV-directed antisense oligodeoxyribonucleotide that turned out to pair with its target RNA at an association rate constant of k=9.2 x 10(4) M(-1) s(-1), which is substantially faster than the annealing rates of artificial antisense RNA so far included in in vitro selection assays.

Computer Simulation↗
PubMed1996

Dissociation of long-chain duplex RNA can occur via strand displacement in vitro: biological implications.

Hammerhead ribozymes with long antisense flanks (>50 bases) have been used successfully to inhibit replication of human immunodeficiency virus type 1 (HIV-1) in living cells. To explain their increased efficacy versus antisense controls or catalytically inactive derivatives, one can consider dissociation of the ribozyme-product complex to allow a complete catalytic cycle. In this work we investigated the dissociation of a double-stranded RNA with 56 bp in vitro. Dissociation was observed in the presence of single-stranded RNA with sequence complementarity to one of the duplex strands. A displacement reaction between RNA single strands and the duplex, but not simple dissociation, was strongly suggested by the concentration dependence of this process, the influence of additional non-complementary sequences on the single strand and by the unusually low Arrhenius activation energy. The strand displacement reaction was slow in vitro at 37 degrees C and physiological ionic strength, but was increased to k approximately 10(3)-10(4)/M/s (approximately 10(4)-fold) at higher temperatures by cetyltrimethylammonium bromide. This compound is thought to enhance non-sequence-specific association of nucleic acids in a mechanistically similar way to that in which cellular hnRNP proteins are thought to act, indicating that strand displacement can be fast and, more importantly, could be tightly regulated in vivo.

Cetrimonium↗
PubMed1996

Kinetic selectivity of complementary nucleic acids: bcr-abl-directed antisense RNA and ribozymes.

Efficacy and sequence specificity are two major requirements in the use of antisense nucleic acids and ribozymes. For long-chain complementary RNA sequences (>30 nt), effects in living cells are correlated with the association rate of the complementary RNA in vitro, but not with the stability of the formed double strand. Thus, sequence selectivity of complementary RNA has to be defined as fast versus slow annealing with the appropriate target or non-target sequences, respectively. In this work, we performed a systematic kinetic analysis to evaluate the selectivity of bcr-abl-directed antisense RNA and hammerhead ribozymes with a length of the complementary sequences of between 20 and 80 bases. By kinetic in vitro selection, we identified oligomeric as well as long-chain complementary RNA that annealed at least tenfold faster with the bcr-abl sequence in comparison with either of the wild-type sequences bcr or abl, respectively. In the presence of selected oligodeoxynucleotide sequences and RNase H, the bcr-abl transcript was specifically hydrolysed out of a mixture containing abl and bcr sequences as well. Hammerhead ribozymes were designed such that binding with their target was facilitated either via helix I or helix III-forming antisense arms but not both. Further, cleavage and binding occurred on opposite sides of the bcr-abl fusion point. Target selectivity was found for a ribozyme that annealed fast via abl sequences and cleaved within the bcr portion of bcr-abl RNA. Kinetic probing and calculations of the local folding potential indicate that the bcr-abl fusion point sequences are not easily accessible for complementary nucleic acids. This study supports the need for more detailed structural investigations of the bcr-abl fusion sequence and forms a more rational basis for the therapeutic use of nucleic acid inhibitors of the aberrant bcr-abl gene expression in Philadelphia chromosome-positive cells.

Base Sequence↗
PubMed1996

Inhibition of constitutive serine phosphatase activity in T lymphoma cells results in phosphorylation of pp19/cofilin and induces apoptosis.

In untransformed T lymphocytes, pp19/cofilin, a cytoplasmic actin-binding protein, undergoes dephosphorylation and nuclear translocation in response to costimulation through accessory receptors (e.g., CD2), but not following TCR/CD3 triggering. In malignant T lymphoma cells, dephosphorylation and nuclear translocation of pp19/cofilin occur spontaneously through constitutive activation of a serine phosphatase. Blockade of these processes by the serine phosphatase inhibitor okadaic acid leads to apoptosis. Moreover, lowering the intracellular pp19/cofilin concentrations by antisense-cofilin transfection results in reduced cloning efficiencies. These findings provide support for the view that pp19/cofilin plays a critical role in the growth and survival of both untransformed and malignant T lymphocytes.

Actin Depolymerizing Factors↗
PubMed1996

In vitro and in vivo action of antisense RNA.

The transient or permanent expression of antisense RNA represents one option to apply antisense techniques in biotechnology and medical research. Despite the increasing importance and use of antisense nucleic acids as well as their significant antisense-specific phenotypic effects in vivo, there is an obvious lack of explanation for the mechanism of their action. By studying naturally occurring antisense RNA and analyzing their mechanism of action we attempt to learn more about the design, the use, and the critical parameters of artificial antisense RNA. Attempts to derive models from biochemical and structural studies for the interactions between antisense RNAs and their targets will be discussed.

Animals↗
PubMed1995

Negative feedback regulation of wild-type p53 biosynthesis.

When growth-arrested mouse fibroblasts re-entered the cell-cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma-irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed-back loop: p53 mRNA has a predicted tendency to form a stable stem-loop structure that involves the 5'-untranslated region (5'-UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5'-UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53-intrinsic RNA re-annealing activity. The inhibition of p53 biosynthesis requires wild-type p53, as it is not observed with MethA mutant p53, p53-catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem-loop structures. Release from negative feed-back regulation of p53 biosynthesis, e.g. after damage-induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell-cycle checkpoint determinant after DNA damage.

3T3 Cells↗