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PubMed2004

Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors.

Using DNA microarrays, we compared global transcript stability profiles following chemical inhibition of transcription to rpb1-1 (a temperature-sensitive allele of yeast RNA polymerase II). Among the five inhibitors tested, the effects of thiolutin and 1,10-phenanthroline were most similar to rpb1-1. A comparison to various microarray data already in the literature revealed similarity between mRNA stability profiles and the transcriptional response to stresses such as heat shock, consistent with the fact that the general stress response includes a transient shutoff of general mRNA transcription. Genes encoding factors involved in rRNA synthesis and ribosome assembly, which are often observed to be coordinately down-regulated in yeast microarray data, were among the least stable transcripts. We examined the effects of deletions of genes encoding deadenylase components Ccr4p and Pan2p and putative RNA-binding proteins Pub1p and Puf4p on the genome-wide pattern of mRNA stability after inhibition of transcription by chemicals and/or heat stress. This examination showed that Ccr4p, the major yeast mRNA deadenylase, contributes to the degradation of transcripts encoding both ribosomal proteins and rRNA synthesis and ribosome assembly factors and mediates a large part of the transcriptional response to heat stress. Pan2p and Puf4p also contributed to the degradation rate of these mRNAs following transcriptional shutoff, while Pub1p preferentially stabilized transcripts encoding ribosomal proteins. Our results indicate that the abundance of ribosome biogenesis factors is controlled at the level of mRNA stability.

Gene Expression Profiling↗
PubMed2002

Three-dimensional finite element analysis of implant stability in the atrophic posterior maxilla: a mathematical study of the sinus floor augmentation.

Sinus lifting is performed with a variety of materials and techniques without a precise knowledge of the quantity of augmentation. This study based on three-dimensional finite element analysis was designed to show which surgical procedure and which amount of peri-implant packing yields the best bony support for dental implants. Eight 3D-FE models were used. Four modeled standard situations simulated quantitatively different packing situations produced by differences in surgical approach: i. no packing; ii. thin 1 mm bony sheath; iii. oblique subcomplete packing; iv. complete bony peri-implant packing up to the implant end. A fifth model compared a standard implant with a length of 13.5 mm and a diameter of 3.75 mm with a 7-mm-long and 5-mm-thick implant. In three additional models the stress response of the bone-implant system was evaluated in the absence of a cortical layer, thus simulating an extreme degree of maxillary atrophy. In all models the modeled implants were loaded at their points of emergence with an assumed force of 100 N. The vector of the loading force was inclined 30 degrees posteriorly relative to the implant axis and 30 degrees away from the sagittal plane. The bone-implant interface was assumed to be perfect simulating full osseointegration. The final evaluation of the FE models showed complete peri-implant packing to reduce displacements of the implant tip by 32% vs. no sheathing/packing. Van Mises' equivalent stresses were used to assess the stresses in both human bone and titanium alloy implants. The highest stress levels in bone were predicted for the case without sufficient implant sheathing. In the models with adequate bony implant support, intrabony stresses were generally reduced by up to - 40%. The structural stiffness of the bone-implant system increased with the extent of sinus floor elevation. The results indicate that more extensive peri-implant packing reduces implant displacement, intrabony stresses and stresses at the bone-implant interface.

Alveolar Ridge Augmentation↗
PubMed1991

Simulation analysis of the stability mutant R96H of T4 lysozyme.

Free energy simulation methods are used to analyze the effects of the mutation Arg 96----His on the stability of T4 lysozyme. The calculated stability change and the lack of significant structural rearrangement in the folded state due to the mutation are in agreement with experimental studies [Kitamura, S., & Sturtevant, J. M. (1989) Biochemistry 28, 3788-3792; Weaver, L. H., et al. (1989) Biochemistry 28, 3793-3797]. By use of thermodynamic integration, the contributions of specific interactions to the free energy change are evaluated. It is shown that a number of contributions that stabilize the wild type or the mutant partially cancel in the overall free energy difference; some of these involve the unfolded state. Comparison of the results with conclusions based on structural and thermodynamic data leads to new insights into the origin of the stability difference between wild-type and mutant proteins. Of particular interest is the importance of the contributions of more distant residues, solvent water, and the covalent linkage of the mutated amino acid. Also, the analysis of the interactions of Arg/His 96 with the C-terminal end of a helix (residues 82-90) makes it clear that the nearby carbonyl groups (Tyr 88 and Asp 89) make the dominant contribution, that the amide groups do not contribute significantly, and that the helix-dipole model is inappropriate for this case.

Arginine↗
PubMedDate not supplied

Static and dynamic roentgenographic analysis of ankle stability in braced and nonbraced stable and functionally unstable ankles.

Patients suffering from functional ankle instability were selected based on a structured interview. Talar tilt was measured using supine ankle stress roentgenographs and standing talar tilt was measured using erect ankle stress roentgenographs. A digital roentgenocinematographic analysis of a 50 degrees ankle sprain simulation was performed to measure dynamic talar tilt and inversion distance between two video images (inversion speed). A significant decrease in pathologic supine talar tilt in unstable ankles was found in the braced compared with the nonbraced situation (talar tilt = 13.1 degrees versus 4.8 degrees with brace). The talar tilt with the brace after activity was still significantly lower than the initial value without the brace. The standing talar tilt of unstable ankles was shown to be significantly lower with the orthosis than without (standing talar tilt = 16.6 degrees versus 12.0 degrees with brace). Roentgenocinematographic evaluation of ankle sprain simulation showed that the mean dynamic talar tilt during simulated sprain decreased significantly in the braced ankles compared with the nonbraced ankles (dynamic talar tilt = 9.8 degrees versus 6.4 degrees braced). A significant decrease in the digital measurement of inversion distance (from 110.6 pixels to 92.4 pixels) was observed in the total sample of 39 ankles during the initial high-speed phase of the simulated sprain. The brace significantly slows down the inversion speed.

Analysis of Variance↗
PubMed2004

Role of serine 10 phosphorylation in p27 stabilization revealed by analysis of p27 knock-in mice harboring a serine 10 mutation.

The inhibition of cyclin-dependent kinase activity by p27 contributes to regulation of cell cycle progression. Serine 10 is the major phosphorylation site of p27, and its phosphorylation has been shown to affect the stability and nuclear export of p27 at the G0-G1 transition in transfected cultured cells. To investigate the physiological relevance of p27 phosphorylation on Ser10, we generated p27 "knock-in" mice that harbor an S10A mutation in this protein. Mice homozygous for the mutation (p27(S10A/S10A) mice) were normal in body size, but the abundance of p27 was decreased in many organs, including brain, thymus, spleen, and testis. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27(S10A/S10A) mice compared with that in wild-type cells, whereas p27 stability in S phase was similar in cells of the two genotypes. The degradation of p27 in cells of the mutant mice at G0 phase was prevented by a proteasome inhibitor. These data indicate that the physiological role of p27 phosphorylation on Ser10 is to stabilize the protein in G0 phase. Unexpectedly, the nuclear export of p27 at the G0-G1 transition occurred normally in p27(S10A/S10A) mouse embryonic fibroblasts, indicating that phosphorylation of Ser10 is dispensable for this process.

Active Transport, Cell Nucleus↗
PubMed2000

Analysis of the stability and degradation products of triptolide.

Triptolide is the major active ingredient of the Chinese herbal remedy Tripterygium wilfordii Hook F. (TwHF). As triptolide content is used to estimate the potency of preparations of TwHF, assessment of its stability is warranted. The accelerated stability of triptolide was investigated in 5% ethanol solution in a light-protected environment at pH 6.9, within a temperature range of 60-90 degrees C. The observed degradation rate followed first-order kinetics. The degradation rate constant (K25 degrees C) obtained by trending line analysis of Arrhenius plots of triptolide was 1.4125 x 10(-4) h(-1). The times to degrade 10% (t1/10) and 50% (t1/2) at 25 degrees C were 31 and 204 days, respectively. Stability tests of triptolide in different solvents and different pH conditions (pH4-10) in a light-protected environment at room temperature demonstrated that basic medium and a hydrophilic solvent were the major factors that accelerated the degradation of triptolide. Triptolide exhibited the fastest degradation rate at pH 10 and the slowest rate at pH 6. In a solvent comparison, triptolide was found to be very stable in chloroform. The stability of triptolide in organic polar solvents tested at both 100% and 90% concentration was greater in ethanol than in methanol than in dimethylsulphoxide. Stability was also greater in a mixture of solvent:pH6 buffer (9:1) than in 100% solvent alone. An exception was ethyl acetate, which is less polar than the other solvents tested, but permitted more rapid degradation of triptolide. Two of the degradation products of triptolide were isolated and identified by HPLC and mass spectroscopy as triptriolide and triptonide. This suggested that the decomposition of triptolide occurred at the C12 and C13 epoxy group and the C14 hydroxyl. The opening of the C12 and C13 epoxy is an irreversible reaction, but the reaction occurring on the C14 hydroxyl is reversible. These results show that the major degradation pathway of triptolide involves decomposition of the C12 and C13 epoxy group. Since this reaction is very slow at 4 degrees C at pH 6, stability is enhanced under these conditions.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Cryptic plasmid pRK2 from Escherichia coli W: sequence analysis and segregational stability.

Cryptic plasmid pRK2 of the strain Escherichia coli W (ATCC 9637), an ancestor of production strains for penicillin G acylase, was sequenced and characterized. Based on the data on replication region and origin (ori sequence AAC, 924-926nt), the plasmid was classified as ColE1-like plasmid. DNA sequence analysis revealed five orfs hypothetical products of which shared a significant sequence similarity with putative proteins encoded by DNA of plasmid pColE1. orf1 codes for protein Rom involved in the control of plasmid replication, orfs 2-5 code for putative mobilization proteins (Mob A-D) that show a high level of similarity with the ones encoded by DNA of plasmids pColE1 and pLG13 (E. coli), pECL18 and pEC01 (Enterobacter cloacae), pSFD10 (Salmonella choleraesuis), and pScol7 (Shigella sonnei). Recombinant plasmids pRS11 (4.91kbp), pRS12 (4.91kbp), pRS2 (2.996kbp), and pRS3 (2.623kbp) that bear the Spectinomycin resistance determinant (Spc(R)) were prepared on the basis of nucleotide sequence of pRK2. These constructs are stably maintained in the population of E. coli cells grown in the absence of the selection pressure for 63 generations. The copy number of Spc(R) constructs in E. coli host grown in antibiotic-free LB medium ranges from 25 to 40 molecules per chromosomal equivalent.

Bacterial Proteins↗
PubMed1993

Mutational analysis of segmental stabilization of transcripts from the Zymomonas mobilis gap-pgk operon.

In Zymomonas mobilis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase are transcribed together from the gap-pgk operon. However, higher levels of the former enzyme are present in the cytoplasm because of increased stability of a 5' segment containing the gap coding region. This segment is bounded by an upstream untranslated region which can be folded into many stem-loop structures and a prominent intercistronic stem-loop. Mutations eliminating a proposed stem-loop in the untranslated region or the intercistronic stem-loop resulted in a decrease in the stability and pool size of the 5' gap segment. Site-specific mutations in the unpaired regions of both of these stems also altered the message pools. Elimination of the intercistronic stem appeared to reduce the endonucleolytic cleavage within the pgk coding region, increasing the stability and abundance of the full-length message. DNA encoding the prominent stem-loop at the 3' end of the message was shown to be a transcriptional terminator both in Z. mobilis and in Escherichia coli. This third stem-loop region (part of the transcriptional terminator) was required to stabilize the full-length gap-pgk message.

Base Sequence↗
PubMed1996

Experimental analysis of functional stability of sagittal split ramus osteotomies secured by miniplates and position screws.

PURPOSE: This study determined the relative functional stabilities of various miniplate systems and configurations used to stabilize sagittal split ramus osteotomies (SSROs) and compared them with conventional internal screw fixation. MATERIALS AND METHODS: The biomechanical model was a reproducible prototype of a mandible sagittal osteotomy with consistent material and geometric properties. After advancing the distal segment by 7 mm, each set of mandible analogs (1 set = 3 analogs) was fixed bilaterally by one of three miniplate systems applied in various configurations, and tested with and without a supplemental 2.4-mm bicortical screw applied in the retromolar region. Reduced analogs were placed in a straining frame, and simulated masticatory loads were applied alternatively to the mandibular first molars. Ensuing osteotomy site displacements were measured by transducers attached to a computer-based data acquisition program. A coordinate transformation procedure was used to convert the component displacements captured by the individual transducers into a common "instability factor" to reflect fixation stability for each construct and loading condition. Instability factors for the individual constructs were compared with each other and with those obtained from analogs reduced exclusively with 2.4-mm position screws. RESULTS: Osteotomies stabilized with a combination of miniplates and position screws were more stable than those stabilized exclusively with miniplates (P < .0001). Post-hoc comparisons of mean instability factors (Dunnet's method) showed the miniplate-position screw combinations to be more stable than the 2.4-mm position screw system used as standard (P < .05). Miniplate systems alone were the least stable of the test constructs, with differential rates of failure between the individual miniplate systems. CONCLUSIONS: Exclusive use of miniplate fixation may not provide the consistent stability necessary for early functional restoration after SSROs. The addition of a position screw in the retromolar region substantially enhances the fixation stability of miniplate systems. The use of miniplates with retromolar position screws offers both technical and stability advantages over conventional miniplate or internal screw fixation. The fixation stability of the miniplate-position screw combination is independent of the type of miniplate system used.

Analysis of Variance↗
PubMed1994

Stability and structural analysis of alpha-amylase from the antarctic psychrophile Alteromonas haloplanctis A23.

The alpha-amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic alpha-amylase. The psychrophilic alpha-amylase is however characterized by a sevenfold higher kcat and kcat/Km values at 4 degrees C and a lower conformational stability estimated as 10 kJ.mol-1 with respect to the porcine enzyme. It is proposed that both properties arise from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low temperatures. This is supported by the fast denaturation rates induced by temperature, urea or guanidinium chloride and by the shift towards low temperatures of the apparent optimal temperature of activity. When compared with the known three-dimensional structure of porcine pancreatic alpha-amylase, homology modelling of the psychrophilic alpha-amylase reveals several features which may be assumed to be responsible for a more flexible, heat-labile conformation: the lack of several surface salt bridges in the (beta/alpha)8 domain, the reduction of the number of weakly polar interactions involving an aromatic side chain, a lower hydrophobicity associated with the increased flexibility index of amino acids forming the hydrophobic clusters and by substitutions of proline for alanine residues in loops connecting secondary structures. The weaker affinity of the enzyme for Ca2+ (Kd = 44 nM) and for Cl- (Kd = 1.2 mM at 4 degrees C) can result from single amino acid substitutions in the Ca(2+)-binding and Cl(-)-binding sites and can also affect the compactness of alpha-amylase.

Amino Acid Sequence↗
PubMed1991

Simulation analysis of the stability mutants R96H of bacteriophage T4 lysozyme and I96A of barnase.

Free energy simulation methods are used to analyse the effects of the mutation Arg-96----His on the stability of bacteriophage T4 lysozyme and of Ile-96----Ala on the stability of barnase. By use of thermodynamic integration, the contributions of specific interactions to the free energy change are evaluated. It is shown that a number of contributions that stabilize the wild-type or the mutant partially cancel in the overall free energy difference; some of these involve the unfolded state. Comparison of the results with conclusions based on structural and thermodynamic data leads to new insights into the origin of the stability difference between wild-type and mutant proteins. For the charged-to-charged amino acid mutation in T4 lysozyme, the importance of the contributions of more distant residues, solvent water and the covalent linkage involving the mutated amino acid are of particular interest. Also, the analysis of the Arg-96 to His mutation with respect to the interactions with the C-terminal end of a helix (residues 82-90) indicates that the nearby carbonyl groups (Tyr-88 and Asp-89) make the dominant contribution, that the amide groups do not contribute significantly and that the helix dipole model is inappropriate for this case. For the non-polar-to-non-polar amino acid mutation in barnase, the solvent contribution is unimportant, and covalent terms are shown to be significant because they do not cancel between the folded and unfolded state.

Bacterial Proteins↗
PubMed2001

Stabilization and linkage analysis of metal-ligated sialic acid containing oligosaccharides.

The dissociation of metal-ligated sialyllactose and sialyl-N-acetyllactosamine was investigated. Metal-ligand derivatization of the carbohydrate samples with the diethylenetriamine ligand and one of four transition metals [Co(II), Ni(II), Cu(II), Zn(II)] suppressed sialic acid loss in the collision-induced dissociation process. Suppression of sialic acid loss allows sialic acid linkage information to be gained through tandem mass spectrometry. Sialic acid stabilization is postulated to occur due to the doubly charged metal ion which allows for deprotonation of the sialic acid moiety. Furthermore, a connection between the metal center and the amount of sialic acid loss was found. These results were rationalized using the Irving-Williams series and a competition between different sites of deprotonation. Analysis of the product ion spectra showed a clear differentiation of sialic acid linkage. Linkage determination is proposed to be effective due to the available conformations allowed by the different linkages. A more flexible linkage will allow more coordination of the sialic acid residue with the metal center, whereas a less flexible linkage will make this interaction unlikely.

Amino Sugars↗
PubMedDate not supplied

Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis.

Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 degrees C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations.

Calorimetry, Differential Scanning↗
PubMed1994

Attachment style, gender, and relationship stability: a longitudinal analysis.

Adult attachment styles in 354 heterosexual couples in serious dating relationships were examined. Principal findings included the following: (a) male and female attachment styles were nonrandomly paired, for example, no anxious-anxious or avoidant-avoidant pairs were found; (b) male and female styles related to concurrent relationship ratings of both partners in different but theoretically meaningful ways; (c) male and female styles contributed significantly to longitudinal prediction of relationship stability and status, even when prior duration and commitment to the relationship were statistically controlled; (d) specifically, relationships of avoidant men and of anxious women were surprisingly stable over 3 years, particularly in light of the relatively poor ratings of these relationships by both partners at Time 1. Discussion focuses on the need to integrate gender role considerations and relationship dynamics and processes into theorizing on adult attachment.

Adult↗
PubMed2005

Closed-loop stability and performance analysis of least-squares and minimum-variance control algorithms for multiconjugate adaptive optics.

Recent progress has been made to compute efficiently the open-loop minimum-variance reconstructor (MVR) for multiconjugate adaptive optics systems by a combination of sparse matrix and iterative techniques. Using spectral analysis, I show that a closed-loop laser guide star multiconjugate adaptive optics control algorithm consisting of MVR cascaded with an integrator control law is unstable. Tosolve this problem, a computationally efficient pseudo-open-loop control (POLC) method was recently proposed. I give a theoretical proof of the stability of this method and demonstrate its superior performance and robustness against misregistration errors compared with conventional least-squares control. This can be accounted for by the fact that POLC incorporates turbulence statistics through its regularization term that can be interpreted as spatial filtering, yielding increased robustness to misregistration. For the Gemini-South 8-m telescope multiconjugate system and for median Cerro Pachon seeing, the performance of POLC in terms of rms wave-front error averaged over a 1-arc min field of view is approximately three times superior to that of a least-squares reconstructor. Performance degradation due to 30% translational misregistration on all three mirrors is approximately a 30% increased rms wave-front error, whereas a least-squares reconstructor is unstable at such a misregistration level.

Journal Article↗
PubMed2005

Analysis of the stability of the spermadhesin PSP-I/PSP-II heterodimer. Effects of Zn2+ and acidic pH.

Spermadhesins are a family of 12-16 kDa proteins with a single CUB domain. PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer. Dimerization markedly affects the binding ability of the subunits. Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface. Pursuing the hypothesis that cryptic binding sites in PSP-I/PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+ and acidic pH on the heterodimer stability. According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma. However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry. The effect is Zn2+-specific and is reversed by EDTA. Destabilization is also observed at acidic pH. Gel filtration analysis using radioiodinated PSP-I/PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+ and acidic pH. Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction. Such a mechanism may be relevant in the regulation of uterine immune reactions.

Acids↗
PubMed1998

In vivo analysis of the stability and fitness of variants recovered from foot-and-mouth disease virus quasispecies.

We have analysed the ability to infect pigs of two foot-and-mouth disease virus (FMDV) variants isolated at low frequencies from virus populations (quasispecies) generated in pigs on infection with a parental virus, C-S8c1. A monoclonal antibody-resistant mutant (MARM21), and a variant isolated at early times post-infection (S-3T1), each exhibiting a unique amino acid substitution in VP1, were able to cause disease in pigs, both by direct inoculation or by contact transmission. The symptoms developed were similar to those produced by C-S8c1 or the related virus C-S15c1. The VP1 sequence of viral RNA directly recovered from lesions of infected animals confirmed the stability of the variant genotypes. Pigs infected with S-3T1 consistently showed an advance of 12 to 24 h in the emergence of fever and lesions when compared to animals infected with C-S8c1 or the remaining variants, an observation consistent with its early isolation. The ability of FMDV variants to compete in vivo with C-S8c1 was investigated in co-infection experiments. Analysis of the proportion of each of the competitors in lesions of co-infected pigs revealed that none of the variants was completely overgrown by the parent. However, co-infection with C-S8c1 and MARM21 resulted in lesions in which C-S8c1 was predominant, indicating a selective disadvantage of this variant in swine. In contrast, lesions from swine co-infected with C-S8c1 and S-3T1 contained similar proportions of the two viruses. These results document fitness variations in vivo among components of the mutant spectrum of FMDV quasispecies.

Animals↗