Diverse antisense mechanisms and applications.
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Animals↗
Biomedical subjects
L Good
Publications and source records attributed to L Good.
Translation repression by antisense sequences.
Antisense sequences that hybridize to messenger RNA can inhibit target gene expression in a variety of ways. The best-known antisense mechanisms trigger messenger RNA (mRNA) degradation; however, translation repression by ribosome interference is a common natural antisense mechanism. In bacteria, there are fascinating examples of cis-encoded and trans-encoded antisense sequences that reversibly repress translation. In eukaryotes, microRNAs that bind 3'UTR target sequences also repress translation, although the mechanism is unclear. An important feature of translation repression is that the mRNA can remain intact during periods of repression, and rapid expression switching can occur in response to cellular and environmental signals. Recent genome analyses indicate many new short noncoding RNAs with predicted antisense activities. Therefore, translation repression by antisense sequences is likely to be a common and important form of posttranscriptional gene control, and such natural mechanisms provide a basis for the development of synthetic antisense gene control in research and drug development.
Animals↗
Tranexamic acid decreases external blood loss but not hidden blood loss in total knee replacement.
BACKGROUND: Total knee arthroplasty (TKA) is often carried out using a tourniquet and shed blood is collected in drains. Tranexamic acid decreases the external blood loss. Some blood loss may be concealed, and the overall effect of tranexamic acid on the haemoglobin (Hb) balance is not known. METHODS: Patients with osteoarthrosis had unilateral cemented TKA using spinal anaesthesia. In a double-blind fashion, they received either placebo (n=24) or tranexamic acid 10 mg kg(-1) (n=27) i.v. just before tourniquet release and 3 h later. The decrease in circulating Hb on the fifth day after surgery, after correction for Hb transfused, was used to calculate the loss of Hb in grams. This value was then expressed as ml of blood loss. RESULTS: The groups had similar characteristics. The median volume of drainage fluid after placebo was 845 (interquartile range 523-990) ml and after tranexamic acid was 385 (331-586) ml (P<0.001). Placebo patients received 2 (0-2) units and tranexamic acid patients 0 (0-0) units of packed red cells (P<0.001). The estimated blood loss was 1426 (1135-1977) ml and 1045 (792-1292) ml, respectively (P<0.001). The hidden loss of blood (calculated as loss minus drainage volume) was 618 (330-1347) ml and 524 (330-9620) ml, respectively (P=0.41). Two patients in each group developed deep vein thrombosis. CONCLUSIONS: Tranexamic acid decreased total blood loss by nearly 30%, drainage volume by approximately 50% and drastically reduced transfusion. However, concealed loss was only marginally influenced by tranexamic acid and was at least as large as the drainage volume.
Aged↗
Ptosis as a remote effect of therapeutic botulinum toxin B injection.
The authors report a patient with cervical dystonia, previously treated with botulinum toxin A (BTX-A), who developed bilateral ptosis and difficulty with accommodation only after botulinum toxin B (BTX-B). High-frequency repetitive nerve stimulation of the abductor digiti minimi demonstrated a 34% increment in compound muscle action potential. No increment in 20 people injected with BTX-A and no cases of ptosis in a chart review of 1,606 BTX-A injections for cervical dystonia were found. The authors conclude that systemic spread of BTX-B can cause symptomatic involvement of autonomic neurons.
Adult↗
Range of motion training in brace vs. plaster immobilization after anterior cruciate ligament reconstruction: a prospective randomized comparison with a 2-year follow-up.
The purpose of this prospective and randomized study was to compare rehabilitation with early range of motion (ROM) training vs immobilization following anterior cruciate ligament (ACL) reconstruction. Fifty patients, undergoing an ACL reconstruction with a bone-patellar tendon-bone graft, were postoperatively allocated randomly to either a plaster cast or a brace for 5 weeks. The brace group had ROM exercises from postoperative day 7. The commencement of ROM exercises was postponed 4 weeks for the plaster group compared to the brace group, but progressed subsequently with equal speed. There was no difference between the groups in the ROM of flexion or extension 20 weeks after the ACL reconstruction and later. Twenty-four months after surgery, the muscle strength deficit in the hamstring muscles (isokinetic measurements; percent difference, injured vs uninjured) was significantly larger in the brace group (mean +/- SD: 5.9 +/- 7.8%, P < 0.01) than in the plaster group (- 0.9 +/- 11.8%, NS) (brace vs plaster group, P < 0.05). Furthermore, there was also a tendency in the brace group to a larger strength deficit in the quadriceps muscle (brace: 11.1 +/- 13.2%, P < 0.001; plaster: 3.8 +/- 12.9%, NS) (brace vs plaster group, P= 0.07). There was no difference between the groups in the total sagittal knee laxity, as measured with an arthrometer, or in the subjective knee function or activity level (Lysholm score together with the Tegner activity level) between the groups. It is concluded that the postoperative treatment with early range of motion training after ACL reconstruction gave as good ROM, knee stability, subjective knee function and activity level as the treatment with immobilization. It is hypothesized that the larger strength deficit observed after rehabilitation with early range of motion training is secondary to the more intensive training and physical therapist involvement that was demanded in order to achieve full ROM following immobilization.
Adolescent↗
Bactericidal antisense effects of peptide-PNA conjugates.
Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli amounting to two orders of magnitude while retaining target specificity. Peptide-PNA conjugates targeted to ribosomal RNA (rRNA) and to messenger RNA (mRNA) encoding the essential fatty acid biosynthesis protein Acp prevented cell growth. The anti-acpP PNA at 2 microM concentration cured HeLa cell cultures noninvasively infected with E. coli K12 without any apparent toxicity to the human cells. These results indicate that peptides can be used to carry antisense PNA agents into bacteria. Such peptide-PNA conjugates open exciting possibilities for anti-infective drug development and provide new tools for microbial genetics.
Amino Acid Sequence↗
Activation of I-kappaB kinase by the HTLV type 1 Tax protein: mechanistic insights into the adaptor function of IKKgamma.
The Tax protein encoded by human T cell leukemia virus type 1 (HTLV-1) induces constitutive nuclear expression of the transcription factor NF-kappaB, causing aberrant expression of a large array of cellular genes. Tax activates NF-kappaB by stimulating the activity of the I-kappaB kinase (IKK), which in turn leads to phosphorylation and degradation of the NF-kappaB inhibitor I-kappaBalpha. In normal T cells, IKK activation occurs transiently on cellular stimulation through the T cell receptor (TCR) and the CD28 costimulatory molecule. However, this inducible kinase is constitutively activated in Tax-expressing and HTLV-1-infected T cells, which contributes to the deregulated nuclear expression of NF-kappaB. As a genetic approach to dissect the pathways mediating IKK activation by Tax and T cell activation signals, somatic cell mutagenesis was performed to isolate signaling-defective mutant Jurkat T cell lines. One of the mutant cell lines was shown to have a defect in NF-kappaB activation by both T cell mitogens and Tax. Interestingly, this mutant cell line lacks expression of the IKK regulatory protein, IKKgamma. Expression of exogenous IKKgamma in the mutant cells restored NF-kappaB activation, thus confirming the essential role of this regulatory factor in IKK activation by the cellular and viral stimuli. Mechanistic studies have shown that Tax physically interacts with IKKgamma via specific domains, including two homologous leucine zipper motifs present in IKKgamma. The Tax/IKKgamma interaction serves to recruit Tax to the IKK catalytic subunits, IKKalpha and IKKbeta, and this recruitment appears to be an essential mechanism by which Tax stimulates the activity of IKK.
Amino Acid Sequence↗
Potent and nontoxic antisense oligonucleotides containing locked nucleic acids.
Insufficient efficacy and/or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a high-affinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA/DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA/DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.
Animals↗
Somatic mutagenesis studies of NF-kappa B signaling in human T cells: evidence for an essential role of IKK gamma in NF-kappa B activation by T-cell costimulatory signals and HTLV-I Tax protein.
NF-kappa B plays a pivotal role in normal T-cell activation and may also mediate human T-cell leukemia virus (HTLV)-induced T-cell transformation. Activation of NF-kappa B by both T-cell costimulatory signals and the HTLV Tax protein involves stimulation of I kappa B kinase (IKK). As a genetic approach to dissect the intermediate steps involved in NF-kappa B activation in human T cells, we performed somatic cell mutagenesis to isolate signaling-defective mutant Jurkat T-cell lines. One of the mutant cell lines was shown to have a specific blockade in the IKK signaling pathway but remained competent in the c-Jun N-terminal kinase and MAP kinase pathways. Interestingly, this mutant cell line lacks expression of IKK gamma, a non-catalytic component of the IKK complex. Expression of exogenous IKK gamma in the mutant cells restored NF-kappa B activation by both the T-cell costimulation agents and Tax. These findings provide genetic evidence for the requirement of IKK gamma in NF-kappa B signaling triggered by both T-cell costimulatory signals and HTLV-I Tax protein.
Antibodies, Monoclonal↗
Essential structural features in the Schizosaccharomyces pombe pre-rRNA 5' external transcribed spacer.
The proximal region in the 5' external transcribed spacer (5'ETS) of the genes encoding ribosomal RNAs in Schizosaccharomyces pombe was examined with respect to structural features which underlie rRNA maturation. Computer analyses and partial digestion with nuclease probes indicate a crucifix-like structure composed primarily of three extended hairpins which are more highly ordered than previously proposed in Saccharomyces cerevisiae. A re-evaluation of the same region in S. cerevisiae indicates a conserved core structure, including the U3 snoRNA binding site within this higher-order structure. The sequences encoding the individual hairpins were deleted by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the effect of these features on rRNA maturation. Quantitative hybridization analyses indicate that the first hairpin only has modest effects on 18 S rRNA maturation, but the other two regions are critical and no mature 18 S rRNA was observed. When smaller changes were systematically introduced into the critical regions, strong correlations were observed with known or putative events in rRNA maturation. Changes associated with an intermediate cleavage site in helix II and with the putative U3 snoRNA binding site were again critical to 18 S rRNA production. In each case, the effects were sequence dependent and not simply the result of disrupted structure. Further analyses of the 5.8 S rRNA indicate that the large ribosomal subunit RNA can be properly processed in each case but the efficiency is reduced by as much as 60 %, an observation which provides new evidence of interdependency in the maturation process. The results illustrate that rRNA processing is more critically dependent on the 5'ETS than previously believed.
Base Sequence↗
Gene expression profiles in HTLV-I-immortalized T cells: deregulated expression of genes involved in apoptosis regulation.
Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia, an acute and often fatal T-cell malignancy. A key step in HTLV-I-induced leukemigenesis is induction of abnormal T-cell growth and survival. Unlike antigen-stimulated T cells, which cease proliferation after a finite number of cell division, HTLV-I-infected T cells proliferate indefinitely (immortalized), thus facilitating occurrence of secondary genetic changes leading to malignant transformation. To explore the molecular basis of HTLV-I-induced abnormal T-cell survival, we compared the gene expression profiles of normal and HTLV-I-immortalized T cells using 'gene array'. These studies revealed a strikingly altered expression pattern of a large number of genes along with HTLV-I-mediated T-cell immortalization. Interestingly, many of these deregulated genes are involved in the control of programmed cell death or apoptosis. These findings indicate that disruption of the cellular apoptosis-regulatory network may play a role in the HTLV-I-mediated oncogenesis.
Apoptosis↗
Calibration and application of an intra-articular force transducer for the measurement of patellar tendon graft forces: an in situ evaluation.
The objective of this study was to evaluate two calibration methods for the "Arthroscopically Implantable Force Probe" (AIFP) that are potentially suitable for in vivo use: (1) a direct, experimentally based method performed by applying a tensile load directly to the graft after it is harvested but prior to implantation (the "pre-implantation" technique), and (2) an indirect method that utilizes cadaver-based analytical expressions to transform the AIFP output versus anterior shear load relationship, which may be established in vivo, to resultant graft load (the "post-implantation" technique). The AIFP outputs during anterior shear loading of the knee joint using these two calibration methods were compared directly to graft force measurements using a ligament cutting protocol and a 6 DOF load cell. The mean percent error (actual-measured)/(actual)* 100) associated with the pre-implantation calibration ranged between 85 and 175 percent, and was dependent on the knee flexion angle tested. The percent error associated with the post-implantation technique was evaluated in two load ranges: loads less than 40 N, and loads greater than 40 N. For graft force values greater than 40 N, the mean percent errors inherent to the post-implantation calibration method ranged between 20 and 29 percent, depending on the knee flexion angle tested. Below 40 N, these errors were substantially greater. Of the two calibration methods evaluated, the post-implantation approach provided a better estimate of the ACL graft force than the pre-implantation technique. However, the errors for the post-implantation approach were still high and suggested that caution should be employed when using implantable force probes for in vivo measurement of ACL graft forces.
Aged↗
Joint position sense is not changed after acute disruption of the anterior cruciate ligament.
We evaluated the impact of acute, isolated ACL disruption on knee joint proprioception by means of passive-active and active-active joint position sense (JPS) measurement techniques. 18 subjects with acute, isolated and unilateral ACL disruption were tested for JPS in a standing position. The test protocol included 6 trials for each leg. In each trial, the lower leg was passively positioned to an index angle approximating either 30 degrees or 70 degrees, followed by 5 active repetitions of the index angle where the subjects attempted to reproduce the index angle to the best of their ability. The errors from the exact index angle reproduction were calculated as both real (showing both magnitude and direction) and absolute values (only magnitude). All subjects had a tendency to reproduce the index angle with both the injured and normal knees in a more flexed position (overestimation). Only the absolute error produced by the active-active test at flexion angles greater than 45 degrees produced a significant difference with a larger error for the normal knee. In all other comparisons between the injured and the normal knee no differences were found. We conclude that the afferent signals which are compromised by an acute tear of the ACL are insignificant compared to afferent signals from the other joint and muscle receptors.
Acute Disease↗
Peptide nucleic acid (PNA) antisense effects in Escherichia coli.
Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake, antisense PNAs may find applications as antimicrobial agents and as tools for microbial functional genomics.
Antisense Elements (Genetics)↗
NF-kappaB-inducing kinase and IkappaB kinase participate in human T-cell leukemia virus I Tax-mediated NF-kappaB activation.
The tax gene product of human T-cell leukemia virus I induces aberrant expression of various cellular genes, which contributes to transformation of host cells. Induction of many Tax target genes is mediated through transcription factor NF-kappaB. Here we show that Tax triggers activation of cellular protein kinases, IkappaB kinase alpha (IKKalpha) and IKKbeta, which phosphorylate the NF-kappaB inhibitory protein IkappaB alpha, resulting in its degradation and NF-kappaB activation. Constitutive IKK activation occurs in both Tax-transfected and human T-cell leukemia virus I-infected T cells. We further demonstrate that Tax-mediated NF-kappaB signaling also requires the NF-kappaB-inducing kinase (NIK). Consistently, inactive forms of either IKKs or NIK attenuate Tax-mediated NF-kappaB activation. Therefore, Tax activates NF-kappaB by targeting cellular signaling molecules, including both IKKs and NIK.
Catalysis↗
Inhibition of translation and bacterial growth by peptide nucleic acid targeted to ribosomal RNA.
Peptide nucleic acid (PNA) is a DNA mimic that has shown considerable promise as a lead compound for developing gene therapeutic drugs. We report that PNAs targeted to functional and accessible sites in ribosomal RNA can inhibit translation in an Escherichia coli cell-free transcription/translation system, with 50% reductions caused by nanomolar PNA concentrations. The effect in vitro is quantitatively similar to that of the known translation inhibitor and antibiotic tetracycline. Also, the targeted PNAs inhibited bacterial growth on agar plates and in liquid culture. A strain of E. coli (AS19) that is more permeable to antibiotics was approximately 10-fold more sensitive to the active PNAs, suggesting that the effect on growth indeed was caused by PNAs that entered cells. Inhibition was not observed when using control PNAs of similar composition but with an unrelated or mismatched sequence. The results demonstrate that ribosomal RNA is a possible target for sequence-designed novel antibiotics based on DNA analogues or mimics.
Base Sequence↗
Antisense inhibition of gene expression in bacteria by PNA targeted to mRNA.
Peptide nucleic acid (PNA) is a DNA mimic with attractive properties for developing improved gene-targeted antisense agents. To test this potential of PNA in bacteria, PNAs were designed to target the start codon regions of the Escherichia coli beta-galactosidase and beta-lactamase genes. Dose-dependent and specific gene inhibition was observed in vitro using low nanomolar PNA concentrations and in vivo using low micromolar concentrations. Inhibition was more efficient for a permeable E. coli strain relative to wild-type K-12. The potency of the anti-beta-lactamase PNAs was abolished by a six base substitution, and inhibition could be re-established using a PNA with compensating base changes. Antisense inhibition of the beta-lactamase gene was sufficient to sensitize resistant cells to the antibiotic ampicillin. The results demonstrate gene- and sequence-specific antisense inhibition in E. coli and open possibilities for antisense antibacterial drugs and gene function analyses in bacteria.
Ampicillin↗
Postural control--a comparison between patients with chronic anterior cruciate ligament insufficiency and healthy individuals.
Postural control in the sagittal plane was evaluated in 22 patients with chronic anterior cruciate ligament (ACL) deficiency and the result was compared to that of a control group of 20 uninjured subjects. Measurement of the body sway was done on a fixed and sway-referenced force plate in both single-limb and two-limb stance, with the eyes open and closed, respectively. Further, an analysis of the postural reactions to perturbations backwards and forwards, respectively, was made in single-limb stance. The results demonstrated statistically significant deficits of the postural control in the patient group compared to the control group, but also within the patient group. There was a significantly higher body sway within the patient group when standing on a stable support surface on the injured limb than standing on the uninjured limb with the eyes open, but no difference with the eyes closed. When standing on a stable support surface, there was a significantly higher body sway in the patient group standing on the injured leg than in the control group, both with eyes open and closed. The patient group also showed a significantly impaired postural control compared to the control group when standing on the uninjured leg with the eyes closed. There was no difference between the groups in the two-limb stance. When standing on the sway-referenced support surface, the patient group had a significantly larger body sway than the control group when the eyes were open, but there was no significant difference between the groups with the eyes closed. The measurement of the postural corrective responses to perturbations backwards and forwards showed that the reaction time measured from the initiation of the force plate translation, and the amplitude of the body sway was significantly greater in the patient group than in the control group. We conclude that patients with a continuing chronic ACL insufficiency several years after injury have an impaired postural control in the antero-posterior direction in single-limb stance on their injured leg. They also show a greater body sway and a prolonged reaction time when subjected to antero-posterior perturbations when standing on their injured leg.
Adolescent↗