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PubMed2000

Gait and postural stability in obese and nonobese prepubertal boys.

OBJECTIVE: To examine differences in gait and postural stability of obese and nonobese prepubertal boys. DESIGN: Percentage of gait cycle in double support was examined to determine significant differences. Postural stability was compared using temporal and frequency measures of the center of pressure in static stance. SETTING: Gait was examined using videography on a 30-meter walkway. Postural stability was examined using a measurement platform. PARTICIPANTS: Ten obese (>95th percentile in body mass index) and 10 nonobese (15th to 90th percentile in body mass index) prepubertal boys aged 8 to 10yrs. INTERVENTION: Participants were examined at three walking cadences as determined by preferred gait cadence. Full vision, darkness, and visually confused conditions were used to accentuate static postural stability differences. MAIN OUTCOME MEASURES: In the presence of dynamic stability differences (gait), static stability measures further investigated stability differences. RESULTS: Obese boys spent significantly (p < .02) greater percentage of gait cycle in dual stance. Obese boys showed significantly (p < .01) greater sway areas, energy, and variability primarily in the medial/lateral direction. CONCLUSIONS: Dual stance differences suggest diminished dynamic stability in obese boys. Greater sway areas in medial/lateral direction in obese boys and the absence of significant frequency measures suggest that the instability observed in obese boys is caused by excess weight rather than underlying postural instability.

Child↗

PubMed2004

Trial-to-trial reproducibility and test-retest stability of two dynamic balance tests among male firefighters.

The aim of this study was to evaluate a dynamic stability test and a functional balance test for trial-to-trial reproducibility and stability over time. In the dynamic stability test a subject stood on the force platform and moved his center of pressure through the targets shown on a computer screen. Functional balance was measured by a test in which the subject had to walk as fast as possible across a wooden plank without falling off. Both tests are repeated six times in two testing periods with an interval of two months. The subjects were 29 male firefighters aged 33 - 56 years. According to intraclass correlation coefficients the functional balance test showed good to excellent (0.78 - 0.96) trial-to-trial reproducibility and stability over time, and the results of the dynamic stability test were moderate (0.16 - 0.81). Individual variation in the repeated tests was considerable. However, the limits of agreement in the functional balance test (+/- 2 - 3 s) and in the performance time of the dynamic stability test (+/- 4 - 5 s) would be reasonable for practical use. Furthermore, the test-retest stability improved when the reliability was estimated from the best of at least three repeated trials with the functional balance test and the best of at least five trials with the dynamic stability test.

Adult↗

PubMed1985

Nature of the charged-group effect on the stability of the C-peptide helix.

The residues responsible for the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A) have been identified by chemical synthesis of analogues and measurement of their helix-forming properties. Each of the residues ionizing between pH 2 and pH 8 has been replaced separately by an uncharged residue. Protonation of Glu-2- is responsible for the sharp decrease in helix stability between pH 5 and pH 2, and deprotonation of His-12+ causes a similar decrease between pH 5 and pH 8. Glu-9- is not needed for helix stability. The results cannot be explained by the Zimm-Bragg model and host-guest data for alpha-helix formation, which predict that the stability of the C-peptide helix should increase when Glu-2- is protonated or when His-12+ is deprotonated. Moreover, histidine+ is a strong helix-breaker in host-guest studies. In proteins, acidic and basic residues tend to occur at opposite ends of alpha-helices: acidic residues occur preferentially near the NH2-terminal end and basic residues near the COOH-terminal end. A possible explanation, based on a helix dipole model, has been given [Blagdon, D. E. & Goodman, M. (1975) Biopolymers 14, 241-245]. Our results are consistent with the helix dipole model and they support the suggestion that the distribution of charged residues in protein helices reflects the helix-stabilizing propensity of those residues. Because Glu-9 is not needed for helix stability, a possible Glu-9-...His-12+ salt bridge does not contribute significantly to helix stability. The role of a possible Glu-2-...Arg-10+ salt bridge has not yet been evaluated. A charged-group effect on alpha-helix stability in water has also been observed in a different peptide system [Ihara, S., Ooi, T. & Takahashi, S. (1982) Biopolymers 21, 131-145]: block copolymers containing (Ala)20 and (Glu)20 show partial helix formation at low temperatures, pH 7.5, where the glutamic acid residues are ionized. (Glu)20(Ala)20Phe forms a helix that is markedly more stable than (Ala)20(Glu)20Phe. The results are consistent with a helix dipole model.

Electrochemistry↗

PubMed1990

Thermodynamic characterization of the stability and the melting behavior of a DNA triplex: a spectroscopic and calorimetric study.

We report a complete thermodynamic characterization of the stability and the melting behavior of an oligomeric DNA triplex. The triplex chosen for study forms by way of major-groove Hoogsteen association of an all-pyrimidine 15-mer single strand (termed y15) with a Watson-Crick 21-mer duplex composed of one purine-rich strand (termed u21) and one pyrimidine-rich strand (termed y21). We find that the near-UV CD spectrum of the triplex can be duplicated by the addition of the B-like CD spectrum of the isolated 21-mer duplex and the CD spectrum of the 15-mer single strand. Spectroscopic and calorimetric measurements show that the triplex (y15.u21.y21) melts by two well-resolved sequential transitions. The first transition (melting temperature, Tm, approximately 30 degrees C) is pH-dependent and involves the thermal expulsion of the 15-mer strand to form the free duplex u21.y21 and the free single strand y15. The second transition (Tm approximately 65 degrees C) is pH-independent between pH 6 and 7 and reflects the thermal disruption of the u21.y21 Watson-Crick duplex to form the component single strands. The thermal stability of the y15.u21.y21 triplex increases with increasing Na+ concentration but is nearly independent of DNA strand concentration. Differential scanning calorimetric measurements at pH 6.5 show the triplex to be enthalpically stabilized by only 2.0 +/- 0.1 kcal/mol of base triplets (1 cal = 4.184 J), whereas the duplex is stabilized by 6.3 +/- 0.3 kcal/mol of base pairs. From the calorimetric data, we calculate that at 25 degrees C the y15.u21.y21 triplex is stabilized by a free energy of only 1.3 +/- 0.1 kcal/mol relative to its component u21.y21 duplex and y15 single strand, whereas the 21-mer duplex is stabilized by a free energy of 17.2 +/- 1.2 kcal/mol relative to its component single strands. The y15 single strand modified by methylation of cytosine at the C-5 position forms a triplex with the u21.y21 duplex, which exhibits enhanced thermal stability. The spectroscopic and calorimetric data reported here provide a quantitative measure of the influence of salt, temperature, pH, strand concentration, and base modification on the stability and the melting behavior of a DNA triplex. Such information should prove useful in designing third-strand oligonucleotides and in defining solution conditions for the effective use of triplex structure formation as a tool for modulating biochemical events.

Base Sequence↗

PubMed1997

Depression of T-cell epitope generation by stabilizing hen lysozyme.

Conformational stability of proteins is an important factor that determines their resistance/susceptibility to proteolytic digestion. Intracellular proteolysis is the key step in antigen presentation events for protein antigens; hence, it is likely that increasing protein stability reduces the antigenicity of proteins. We prepared three hen egg white lysozyme derivatives possessing different stabilities by chemical modification to clarify the relationship between conformational stability and the antigenicity of the protein. One of the derivatives was conformationally unstabilized by removing one intramolecular disulfide bond, whereas the two others were stabilized by the addition of an intramolecular cross-link. The antigenicity of these derivatives was evaluated using hen egg white lysozyme-specific T-cell hybridoma cells and a B-lymphoma cell line, A20, as antigen-presenting cells. With an increase in conformational stability, the T-cell response decreased. However, the reduction was not derived from the inefficiency of internalization to A20 cells nor the alteration of antigenicity by chemical modifications. Moreover, from analyses of their susceptibility to proteolysis and the kinetics of presentation of the T-cell epitope, it was confirmed that increasing protein stability led to the depression of T-cell epitope generation by increasing resistance to proteolysis. These results have an important implication in devising a new strategy for manipulating T-cell response by the stability of protein antigen.

Animals↗

PubMed1998

Nerve growth factor regulation of m4 muscarinic receptor mRNA stability but not gene transcription requires mitogen-activated protein kinase activity.

Nerve growth factor (NGF) up-regulated steady-state levels of m4 muscarinic acetylcholine receptor (mAChR) mRNA in PC12 cells. Up-regulation of mRNA levels was associated with a corresponding increase in mAChR binding sites. Two other growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), up-regulated m4 mRNA and mAChR binding sites. Treatment of PC12 cells with NGF and bFGF, but not EGF, has previously been demonstrated to result in sustained activation of mitogen-activated protein kinase (MAPK). Analogously, NGF and bFGF, but not EGF, increased the stability of m4 mRNA in PC12 cells. In HER-PC12 cells, a clonal PC12 cell transfectant overexpressing EGF receptors and displaying sustained MAPK activation upon receptor stimulation, EGF treatment stabilized the m4 transcript. A synthetic inhibitor of MAPK kinase, PD98059, inhibited growth factor-induced stabilization of the m4 transcript in both PC12 and HER-PC12 cells. These findings demonstrate that the MAPK pathway is involved in transcript stabilization. Cycloheximide pretreatment abolished the post-transcriptional effect of NGF, indicating that de novo protein synthesis was required for the observed increase in m4 mRNA stability. By contrast, cycloheximide had no discernible post-transcriptional effect if added after NGF treatment, suggesting that an inducible yet stable protein factor was involved in m4 mRNA decay. An unusually well conserved 137 nucleotides of m4 3'-untranslated region has been identified by sequence comparison with other mRNAs that are post-transcriptionally regulated by NGF. In PC12 cells that heterologously overexpress this region, we demonstrate that NGF no longer stabilizes endogenous m4 mRNA. This conserved region probably represents an NGF-responsive element involved in mRNA stability regulation. Finally, transcription of the m4 gene can be induced by all three growth factors but is not dependent on MAPK activity, unlike growth factor-induced m4 mRNA stabilization.

Animals↗

PubMed2003

Regulation of cyclooxgenase-2 mRNA stability by taxanes: evidence for involvement of p38, MAPKAPK-2, and HuR.

Taxanes are widely used to treat malignancies and are known to modulate the transcription of several genes. We investigated the effects of taxanes (docetaxel and paclitaxel) on cyclooxygenase-2 (COX-2) transcription and mRNA stability in human mammary epithelial cells. As reported previously for paclitaxel, docetaxel stimulated COX-2 transcription by an AP-1-dependent mechanism. Treatment with taxanes also enhanced the stability of COX-2 mRNA. To define the mechanism by which taxanes stabilized COX-2 mRNA, transient transfections were carried out using luciferase expression constructs containing the COX-2 3'-untranslated region (3'-untranslated region (UTR)). The stabilizing effects of taxanes were localized to the AU-rich region of COX-2 3'-UTR. RNA binding studies indicated that taxanes stimulated the binding of HuR to the AU-rich region of the COX-2 3'-UTR. Overexpression of antisense HuR suppressed taxane-mediated induction of COX-2 3'-UTR activity. We next investigated the signal transduction pathway responsible for taxane-mediated induction of COX-2. Taxanes enhanced protein kinase C activity; overexpressing dominant negative PKC-alpha suppressed taxane-mediated stimulation of both COX-2 3'-UTR and 5'-promoter activities. Interestingly, ERK1/2, JNK, and p38 MAPKs were important for taxane-mediated activation of COX-2 transcription, but only p38 MAPK appeared to be responsible for the increase in COX-2 mRNA stability. MAPKAPK-2, a known target of p38 MAPK, contributed to increased COX-2 mRNA stability following taxane treatment. SB 202190, a selective p38 MAPK inhibitor, and dexamethasone suppressed taxane-mediated stimulation of the COX-2 3'-UTR and binding of HuR. Taken together, these data indicate that taxanes induce COX-2 by stimulating both transcription and mRNA stability. To the best of our knowledge, this is the first evidence that taxanes can promote stabilization of mRNA in addition to modulating gene transcription.

Antigens, Surface↗

PubMed2004

Regulation of unsaturated fatty acid biosynthesis in Saccharomyces: the endoplasmic reticulum membrane protein, Mga2p, a transcription activator of the OLE1 gene, regulates the stability of the OLE1 mRNA through exosome-mediated mechanisms.

The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta9 fatty-acid desaturase, whose expression is regulated through transcriptional and mRNA stability controls. In wild type cells grown on fatty acid-free medium, OLE1 mRNA has a half-life of 10 +/- 1.5 min (basal stability) that becomes highly unstable when cells are exposed to unsaturated fatty acids (regulated stability). Activation of OLE1 transcription is dependent on N-terminal fragments of two membrane proteins, Mga2p and Spt23p, that are proteolytically released from the membrane by a ubiquitin-mediated mechanism. Surprisingly, disruption of the MGA2 gene also reduces the half-life of the OLE1 transcript and abolishes fatty acid regulated instability. Disruption of its cognate, SPT23, has no effect on the half-life of the mRNA. Mga2p appears to have two distinct functions with respect to the OLE1 mRNA stability: a stabilizing effect in cells grown in fatty acid-free medium and a destabilizing function in cells that are exposed to unsaturated fatty acids. These functions are independent of OLE1 transcription and can confer basal and regulated stability on OLE1 mRNAs that are produced under the control of the unrelated GAL1 promoter. Expression of soluble, N-terminal fragments of Mga2p stabilize the transcript but do not confer fatty acid-regulated instability on the mRNA suggesting that the stabilizing functions of Mga2p do not require membrane processing and that modifications to the protein introduced during proteolysis may play a role in the destabilizing effect. An analysis of mutants that are defective in mRNA degradation indicate that the Mga2p-requiring control mechanism that regulates the fatty acid-mediated instability of the OLE1 transcript acts by activating exosomal 3' --> 5'-exonuclease degradation activity.

Cell Membrane↗

PubMed2005

Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA subunits and AKAP 95 from RSW extracts by immunoprecipitation resulted in a marked loss of mRNA stabilization activity indicating that the presence of the PKA regulatory and catalytic subunits as well as AKAP 95 in the CSR-protein complexes was absolutely necessary to achieve LDH-A mRNA stabilization.

3' Untranslated Regions↗

PubMed2006

Cholesterol precursors stabilize ordinary and ceramide-rich ordered lipid domains (lipid rafts) to different degrees. Implications for the Bloch hypothesis and sterol biosynthesis disorders.

Genetic disorders of cholesterol biosynthesis result in accumulation of cholesterol precursors and cause severe disease. We examined whether cholesterol precursors alter the stability and properties of ordered lipid domains (rafts). Tempo quenching of a raft-binding fluorophore was used to measure raft stability in vesicles containing sterol, dioleoylphosphatidylcholine, and one of the following ordered domain-forming lipids/lipid mixtures: dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), a SM/cerebroside mixture or a SM/ceramide (cer) mixture. Relative to cholesterol, early cholesterol precursors containing an 8-9 double bond (lanosterol, dihydrolanosterol, zymosterol, and zymostenol) only weakly stabilized raft formation by SM or DPPC. Desmosterol, a late precursor containing the same 5-6 double bond as cholesterol, but with an additional 24-25 double bond, also stabilized domain formation weakly. In contrast, two late precursors containing 7-8 double bonds (lathosterol and 7-dehydrocholesterol) were better raft stabilizers than cholesterol. For vesicles containing SM/cerebroside and SM/cer mixtures the effect of precursor upon raft stability was small, although the relative effects of different precursors were the same. Using both detergent resistance and a novel assay involving fluorescence quenching induced by certain sterols we found cholesterol precursors were displaced from cer-rich rafts, and could displace cer from rafts. Precursor displacement by cer was inversely correlated to precursor raft-stabilizing abilities, whereas precursor displacement of cer was greatest for the most highly raft-stabilizing precursors. These observations support the hypothesis that sterols and cer compete for raft-association (Megha, and London, E. (2004) J. Biol. Chem. 279, 9997-10004). The results of this study have important implications for how precursors might alter raft structure and function in cells, and for the Bloch hypothesis, which postulates that sterol properties are gradually optimized for function along the biosynthetic pathway.

Ceramides↗

PubMed2004

A divergent canonical WNT-signaling pathway regulates microtubule dynamics: dishevelled signals locally to stabilize microtubules.

Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3beta (GSK-3beta). In the canonical WNT pathway, the negative regulator Axin forms a complex with beta-catenin and GSK-3beta, resulting in beta-catenin degradation. Inhibition of GSK-3beta by DVL increases beta-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3beta through a transcription- and beta-catenin-independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3beta-mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3beta, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability.

Adaptor Proteins, Signal Transducing↗

PubMed2025

G4STAB: a multi-input deep learning model to predict G-quadruplex thermodynamic stability based on sequence and salt concentration.

MOTIVATION: G-quadruplexes (G4s) are non-canonical nucleic acid structures formed in guanine-rich regions that modulate gene regulation and genomic stability. The thermodynamic stability of G4s directly influences their biological functions and potential as therapeutic targets. However, current quantitative frameworks for predicting G4 stability rely on predetermined structural features, limiting their effectiveness for diverse G4 topologies, and fail to account for environmental factors such as ion concentration and pH that significantly modulate G4 stability in cellular contexts. RESULTS: We present G4STAB, a multi-input deep learning neural network that accurately predicts DNA G4 melting temperatures based on sequence features, salt concentration, and pH. Trained on 2382 diverse DNA G4 sequences, our model achieves high accuracy (R 2=0.8) without relying on predetermined G4 structural features. G4STAB successfully captures established G4 stability determinants and proposes previously unobserved sequence-stability relationships. Analysis of 391 502 experimentally validated G4s reveals that cancer-like ionic environments alter G4 stability profiles, with a 13.5-fold increase in the number of structures exhibiting physiological melting temperatures (36-42°C). These findings suggest systematic genomic patterns in G4 stability responses across chromosomes and gene types. AVAILABILITY AND IMPLEMENTATION: G4STAB is available at https://github.com/donn-liew/G4STAB; G4STAB web database interface is available at https://donn-liew.github.io/g4stab-web-database/.

G-Quadruplexes↗

PubMed2002

Deficits and recovery of head and trunk orientation and stabilization after unilateral vestibular loss.

The aim of the study was to analyse changes in the orientation and stabilization of the head and trunk and their recovery after complete unilateral loss of vestibular information in humans. The ability of nine Ménière's patients to orient and stabilize their heads and trunks in space was investigated during a simple dynamic task of knee-bends and compared with the performance of 10 healthy subjects. Patients' performance was recorded before unilateral vestibular neurotomy (UVN) and during the time-course of recovery (1 week, 1 month, 3 months). Experiments were performed both in eyes open (EO) and eyes closed (EC) conditions to evaluate the role of visual cues in the recovery process. Head and trunk mean angular position (orientation) and mean maximal angular rotation (stabilization) in the roll plane and the yaw plane were recorded using a video motion analysis system. The results indicate that, in the acute stage after UVN (1 week), patients exhibit marked impairments in head and trunk orientation in both visual conditions. In the EC condition, head and trunk were deviated towards the operated side in the roll plane and the yaw plane. Head and trunk stabilization in space was impaired in the roll plane and associated with increased stabilization of the head on the shoulders. Interestingly, vision caused a complete inversion of the orientation pattern, with head and trunk rotations towards the intact side in the roll plane and the yaw plane. Relative to darkness, vision also reduced head and trunk oscillations. Recovery from abnormal head orientation in the light and impaired head stability in both visual conditions was achieved within 1 month and 3 months after UVN, respectively. However, head and trunk orientation in the dark and trunk stabilization in the roll plane remained uncompensated 3 months post-lesion. These results suggest that unilateral vestibular loss leads to a postural syndrome similar to that described previously for various animal species. They confirm the necessity of vestibular inputs for properly stabilizing head and trunk during self-generated displacements in healthy subjects. They also support the notion that vestibular compensation relies on visual cues whose substitution role gradually decreases after UVN.

Adaptation, Physiological↗

PubMed2002

Joint effects of pleiotropic selection and stabilizing selection on the maintenance of quantitative genetic variation at mutation-selection balance.

In quantitative genetics, there are two basic "conflicting" observations: abundant polygenic variation and strong stabilizing selection that should rapidly deplete that variation. This conflict, although having attracted much theoretical attention, still stands open. Two classes of model have been proposed: real stabilizing selection directly on the metric trait under study and apparent stabilizing selection caused solely by the deleterious pleiotropic side effects of mutations on fitness. Here these models are combined and the total stabilizing selection observed is assumed to derive simultaneously through these two different mechanisms. Mutations have effects on a metric trait and on fitness, and both effects vary continuously. The genetic variance (V(G)) and the observed strength of total stabilizing selection (V(s,t)) are analyzed with a rare-alleles model. Both kinds of selection reduce V(G) but their roles in depleting it are not independent: The magnitude of pleiotropic selection depends on real stabilizing selection and such dependence is subject to the shape of the distributions of mutational effects. The genetic variation maintained thus depends on the kurtosis as well as the variance of mutational effects: All else being equal, V(G) increases with increasing leptokurtosis of mutational effects on fitness, while for a given distribution of mutational effects on fitness, V(G) decreases with increasing leptokurtosis of mutational effects on the trait. The V(G) and V(s,t) are determined primarily by real stabilizing selection while pleiotropic effects, which can be large, have only a limited impact. This finding provides some promise that a high heritability can be explained under strong total stabilizing selection for what are regarded as typical values of mutation and selection parameters.

Genetic Variation↗

PubMed1993

Accelerated poly(A) loss and mRNA stabilization are independent effects of protein synthesis inhibition on alpha-tubulin mRNA in Chlamydomonas.

In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.

Animals↗

PubMed1996

Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.

Eight of the nine homeobox genes of the Hoxb locus encode proteins which contain a conserved hexapeptide motif upstream from the homeodomain. All eight proteins (Hoxb-1-Hoxb-8) bind to a target oligonucleotide in the presence of Pbx1a under conditions where minimal or no binding is detected for the Hox or Pbx1a proteins alone. The stabilities of the Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the middle of the locus (Hoxb-5 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stability and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-3) form complexes which are very unstable. Although Hox-b proteins containing longer linker sequences between the hexapeptide and homeodomains formed unstable complexes, shortening the linker did not confer complex stability. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variations within the hexapeptides of specific Hox proteins also do not explain complex stability differences. However, two core amino acids (tryptophan and methionine) which are absolutely conserved within the hexapeptide domains appear to be required for complex formation. Removal of N- and C-terminal flanking regions did not influence complex stability and the members of paralog group 4 (Hoxa-4, b-4, c-4 and d-4), which share highly conserved hexapeptides, linkers and homeodomains but different flanking regions, form complexes of similar stability. These data suggest that the structural features of Hox proteins which determine Hox-Pbx1a-DNA complex stability reside within the precise structural relationships between the homeodomain, hexapeptide and linker regions.

Amino Acid Sequence↗

PubMed2003

Rational combination of strategies to achieve synergistic stabilization of triplex.

The combination of two stabilizing strategies to increase stability of nucleic acid assembly is not always resulted in synergistic effect. In the present study, to explore the rational way to select the combination for synergistically stabilizing strategies, we examined the kinetic effects of different triplex-stabilizing strategies, poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO). The former increased the association rate constant, whereas the latter decreased the dissociation rate constant in triplex equilibrium. Each strategy increased the binding constant of the triplex formation by nearly two orders of magnitude. The combination of both stabilizing strategies, which was the triplex formation with PN TFO in the presence of the copolymer, increased the binding constant by nearly four orders of magnitude, implying successful synergy of their activities. The kinetically orchestrated effects in which the copolymer and the PN modification contribute to distinct ingredients in triplex equilibrium achieved the observed synergistic stabilization. We conclude that kinetic analyses of stabilizing effects enable us to select a rational combination of stabilizing strategies.

Base Sequence↗

PubMed1999

Experimental verification of the 'stability profile of mutant protein' (SPMP) data using mutant human lysozymes.

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishikawa,K., 1995, J. Mol. Biol., 248, 733-738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudo-energy potential developed for evaluating structure-sequence compatibility in the structure prediction method, the 3D-1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability of SPMP. The thermodynamic parameters for denaturation and crystal structures of these mutant proteins were determined. One mutant protein was stabilized as expected, compared with the wild-type protein. However, the others were not stabilized even though the structural changes were subtle, indicating that SPMP overestimates the increase in stability or underestimates negative effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP. The correlation of the stability changes between the experiment and prediction depended on the types of substitution: there were some correlations for proline mutants and cavity-creating mutants, but no correlation for mutants related to side-chain hydrogen bonds. The present results may indicate some additional factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.

Amino Acid Substitution↗