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D L Sokol

Publications and source records attributed to D L Sokol.

9 recordsLinked to original sources
PubMed1999

Monitoring antisense oligodeoxynucleotide activity in hematopoietic cells.

Traditionally, methods designed to impair translation through direct interactions with target messenger RNA (mRNA) have been designated as "antisense" strategies because of their reliance on the formation of reverse complementary (antisense) Watson-Crick base pairs between the targeting oligodeoxynucleotide (ODN) and the mRNA whose function is to be disrupted. Proof of putative "antisense effects," and other mechanistic studies, would be greatly facilitated by the ability to directly demonstrate hybridization between an antisense (AS) ODN and its mRNA target in vivo. In addition, evidence of AS activity by demonstrating reduced levels of RNA or protein or by showing cleaved target molecules would lend proof of the concept. In this article we discuss how AS ODN may be used to down-regulate target gene expression with an emphasis on those targets chosen for our investigations, and we summarize the methods employed for this type of study.

Animals↗
PubMed1998

Real time detection of DNA.RNA hybridization in living cells.

Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized "molecular beacon" (MB) reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5' and 3' ends. In the absence of a complementary nucleic acid strand, the MB remains in a stem-loop conformation where fluorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. Solution hybridization studies revealed that in the presence of a complementary strand targeted MB could yield up to a 60-fold increase in fluorescence intensity in comparison to control MB. By using a fluorescence microscope fitted with UV fluoride lenses, the detection limit of preformed MB/target sequence duplexes microinjected into cells was found to be >/=1 x 10(-1) ag of MB, or approximately 10 molecules of mRNA. On the basis of this exquisite sensitivity, real-time detection of MB/target mRNA hybridization in living cells was attempted by microinjecting MB targeted to the vav protooncogene, or control MB, into K562 human leukemia cells. Within 15 min, confocal microscopy revealed fluorescence in cells injected with targeted, but not control, MB. These studies suggest that real-time visualization and localization of oligonucleotide/mRNA interactions is now possible. MB could find utility in studying RNA processing, trafficking, and folding in living cells. We hypothesize that MB may also prove useful for finding targetable mRNA sequence under physiologic conditions.

Animals↗
PubMed1998

Regulation of CAT protein by ribozyme and antisense mRNA in transgenic mice.

Transgenic mouse lines were engineered to express stably antisense mRNA or antisense mRNA containing catalytic ribozyme (rbz) structures complementary to bacterial chloramphenicol acetyltransferase (CAT) gene transcripts. One transgenic line expressed antisense mRNA that specifically targeted full-length CAT coding sequences (ACAT). Another transgenic line expressed full-length antisense CAT mRNA which was modified by mutagensis to include four rbz cassettes (rbz-ACAT) in order to compare antisense versus antisense-rbz function in vivo. Preliminary data were also collected from a transgenic mouse line expressing antisense mRNA targeting 72% of the 5' region of CAT coding sequences (5' ACAT). All constructs contained similar control elements in their design. Promoter elements were derived from the bovine alpha s1-casein gene, while the small t intron and 3' control sequences were derived from SV40. The ability of these various constructs to down-regulate CAT protein levels was compared by analysis of CAT protein production in lactating double-hemizygous transgenic female mice. Every double-hemizygous mouse analysed expressed mRNA from the alpha s1-casein-CAT construct (Clarke et al., 1994) and equivalent levels of mRNA from one of the three antisense constructs. Transgenic mouse lines expressing both ACAT and CAT mRNA down-regulated CAT protein levels by 90% of that found in the CAT only transgenic population. Similarly, double-hemizygous transgenic lines expressing both rbz-ACAT and CAT mRNA regulated CAT protein levels by 87%. Preliminary data suggests that expression of mRNA from 5' ACAT/CAT double-hemizygote mice allowed approximately 67% down-regulation of normal CAT protein levels. We conclude that incorporation of multiple ribozymes within the full-length antisense CAT construct does not enhance the effectiveness of antisense mRNA in the down-regulation of CAT protein production in our system.

Animals↗
PubMed1996

Antisense and ribozyme constructs in transgenic animals.

Geneticists have long sought the ability to add or subtract individual genes from an organism's genome, or to be able to alter the level of expression of a gene in a targeted, developmentally and tissue-specific manner. The development of transgenic technology realized the possibilities of increasing the expression of a specific gene or the transfer of a new gene into an animal. Homologous recombination techniques allow the deletion or alteration of a gene in vivo. The production of transgenic animals incorporating a gene construct that expresses a complimentary antisense RNA to a targeted gene, or an antisense RNA incorporating a catalytic, ribozyme sequence, have been suggested as a potential mechanism for obtaining the developmentally and tissue-specific down-regulation of expression of a targeted gene in vivo. In this paper we review the current literature with respect to the application of antisense and ribozyme constructs in transgenic animals and conclude that such constructs can effectively downregulate the level of mRNA from a target gene, the amount of protein produced in the cell, and result in phenotypic consequences.

Animals↗
PubMed1996

Gene therapy: basic concepts and recent advances.

After years of preliminary in vitro and in vivo modeling, the first human gene therapy clinical trial was initiated approximately 5 years ago. The experience gained from this trial, and the others that have come after it, has shown that effective and efficient human gene therapy remains a tantalizing but elusive goal. Nevertheless, interest in this area of therapeutics continues to grow and over 100 gene therapy protocols have now been submitted to the Recombinant DNA Advisory Committee ("RAC") of the National Institutes of Health for approval. The goal of these protocols vary, but include correction of inherited gene defects, engineering new traits into cells such as diminished chemosensitivity in the case of bone marrow cells, or, in the case of tumor cells, expression of new immunophenotypes. Marking cells with a gene whose function can easily be detected, thereby rendering the cell traceable in vitro and in vivo is also the goal of several clinical trials. At the same time, different means of gene transfer are been investigated for the efficiency with which they deliver "therapeutic nucleotides" into their cellular targets. In this review, we recount some of the major developments that have occurred in these various areas and discuss their impact on progress in the field.

Animals↗
PubMed1995

Expression and physiologic significance of Kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34+, c-Kit+ marrow cells.

To determine the potential role of autocrine growth factor production in regulating primitive human hematopoietic cell development, we examined highly purified CD34+, c-Kit+ marrow mononuclear cells for expression of c-Kit ligand (KL) and stem cell tyrosine kinase 1 (stk1) ligand (STK1-L). Normal marrow mononuclear cells coexpressing CD34 and c-Kit were isolated by a combination of immunomagnetic bead isolation and fluorescence-activated cell sorting. Purified cells were then screened for expression of KL and stk1-L mRNA using a sensitive reverse transcription-polymerase chain reaction method. Using this approach, expression of both cytokine genes at the mRNA level was found in this highly enriched cell population. We then examined the functional significance of these mRNAs by inhibiting their expression with antisense (AS) oligodeoxynucleotides (ODN). In comparison to untreated or control ODN treated cells, inhibition of KL led to a 70% and 89% inhibition in burst-forming unit-erythroid (BFU-E) and colony-forming unit-Mix (CFU-Mix) colonies but had no significant effect on CFU-granulocyte-macrophage (CFU-GM) cloning efficiency. In contrast, inhibition of STK1-L alone had no effect on colony formation. However, when STK1-L AS ODN was combined with KL AS ODN, additive inhibition of CFU-GM and CFU-MIX but not of BFU-E colonies was observed. These findings, along with those of our previous studies showing inhibition of primitive hematopoietic cell growth with antisense ODN directed towards the stk1 receptor, suggest the possibility that both receptor/ligand axes regulate primitive hematopoietic cell growth via an autocrine growth loop.

Antigens, CD↗
PubMed1995

Vav is necessary for prolactin-stimulated proliferation and is translocated into the nucleus of a T-cell line.

Stimulation of the prolactin receptor (PRLr) with ligand activates multiple kinase cascades. The proximal mediators involved in the activation of the PRL-activated Raf-1 cascade in T-cells, however, remain poorly characterized. The role of one proximal signaling protein, namely p95vav, during PRLr signal transduction was examined in the Nb2 T-cell line. The novel results obtained here indicate that Vav is transiently associated with the PRLr and is necessary for PRL-stimulated proliferation. During PRL stimulation, a rapid and dramatic increase in guanine nucleotide exchange factor (GEF) activity was found to be associated with Vav immunoprecipitates. Concomitantly, an increase in Vav phosphorylation on serine-threonine residues was observed. The Vav-associated GEF activation could be inhibited by staurosporine and calphostin, but not herbimycin, suggesting a modulatory role for phosphorylation at serine-threonine residues. Treatment of Nb2 cells with antisense Vav oligonucleotide ablated Vav expression and blocked PRL-driven proliferation, but failed to inhibit PRL-induced GEF activation within Nb2 lysates. These data indicate that GEF activity may not be intrinsic to Vav as has been previously suggested, but either resides in or is complemented by an associated GEF. Subsequent to the transient activation of associated GEF activity, Vav was found to translocate into the Nb2 cell nucleus. Thus, Vav may utilize two independent mechanisms in T-cells, namely the activation of an associated GEF and direct nuclear internalization, to mediate PRLr signaling.

Animals↗
PubMed1989

Flexibility characteristics of elite female and male volleyball players.

It is a common assumption that increased range of motion is related to high level performance, however it is difficult to provide conclusive evidence for this assumption due to the many variables contributing to skilled performance. The purpose of this study was to compare shoulder and hip flexibility to jumping height of members of the men's and women's United States Olympic Festival volleyball teams. Twenty-four men and 22 women were measured for standing vertical jump (SVJ) and approach vertical jump (AVJ), as well as range of shoulder extension (SHO) and hip flexion (HIP). A significant and positive correlation was revealed between AVJ and HIP for the men r = 0.42, p less than 0.03). For the women, significant and negative correlations resulted between SVJ and HIP (r = -0.54, p = 0.009) and between AVJ and HIP (r = -0.47, p = 0.03). Although a positive correlation between flexibility and AVJ observed for the men supported the assumption that greater flexibility is related to greater skilled performance, the opposite results occurred for the women. The negative correlations for the women indicate that the players with the greatest vertical jumps had the least hip flexibility. It was concluded that greater hip flexibility may benefit men more than women for jumping ability.

Biomechanical Phenomena↗