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Global analysis of mRNA stability in the archaeon Sulfolobus.

BACKGROUND: Transcript half-lives differ between organisms, and between groups of genes within the same organism. The mechanisms underlying these differences are not clear, nor are the biochemical properties that determine the stability of a transcript. To address these issues, genome-wide mRNA decay studies have been conducted in eukaryotes and bacteria. In contrast, relatively little is known about RNA stability in the third domain of life, Archaea. Here, we present a microarray-based analysis of mRNA half-lives in the hyperthermophilic crenarchaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, constituting the first genome-wide study of RNA decay in archaea. RESULTS: The two transcriptomes displayed similar half-life distributions, with medians of about five minutes. Growth-related genes, such as those involved in transcription, translation and energy production, were over-represented among unstable transcripts, whereas uncharacterized genes were over-represented among the most stable. Half-life was negatively correlated with transcript abundance and, unlike the situation in other organisms, also negatively correlated with transcript length. CONCLUSION: The mRNA half-life distribution of Sulfolobus species is similar to those of much faster growing bacteria, contrasting with the earlier observation that median mRNA half-life is proportional to the minimal length of the cell cycle. Instead, short half-lives may be a general feature of prokaryotic transcriptomes, possibly related to the absence of a nucleus and/or more limited post-transcriptional regulatory mechanisms. The pattern of growth-related transcripts being among the least stable in Sulfolobus may also indicate that the short half-lives reflect a necessity to rapidly reprogram gene expression upon sudden changes in environmental conditions.

DNA, Archaeal↗

Phase-plane analysis of gaze stabilization to high acceleration head thrusts: a continuum across normal subjects and patients with loss of vestibular function.

We investigated the vestibulo-ocular reflex (VOR) during high-acceleration, yaw-axis, head rotations in 12 normals and 15 patients with vestibular loss [7 unilateral vestibular deficient (UVD) and 8 bilateral vestibular deficient (BVD)]. We analyzed gaze stabilization within a 200-ms window after head rotation began, using phase planes, which allowed simultaneous analysis of gaze velocity and gaze position. These "gaze planes" revealed critical dynamic information not easily gleaned from traditional gain measurements. We found linear relationships between peak gaze-velocity and peak gaze-position error when normalized to peak head speed and position, respectively. Values fell on a continuum, increasing from normals, to normals tested with very high acceleration (VHA = 10,000-20,000 degrees/s2), to UVD patients during rotations toward the intact side, to UVD patients during rotations toward the lesioned side, to BVD patients. We classified compensatory gaze corrections as gaze-position corrections (GPCs) or gaze-velocity error corrections (GVCs). We defined patients as better-compensated when the value of their end gaze position was low relative to peak gaze position. In the gaze plane this criterion corresponded to relatively stereotyped patterns over many rotations, and appearance of high velocity (100-400 degrees/s) GPCs in the gaze plane ending quadrant (150-200 ms after head movement onset). In less-compensated patients, and normals at VHA, more GVCs were generated, and GPCs were generated only after gaze-velocity error was minimized. These findings suggest that challenges to compensatory vestibular function can be from vestibular deficiency or novel stimuli not previously experienced. Similar patterns of challenge and compensation were observed in both patients with vestibular loss and normal subjects.

Acceleration↗

Analysis and microbiological stability of an isosorbide 50% mixture.

Isosorbide mixture containing 50% w/v isosorbide and 5% w/v sorbitol is used in the treatment of hydrocephalus. The G.L.C. analysis of the mixture is described and its antimicrobial properties investigated. Results for the determination of lead in the mixture are given and the significance of the levels discussed with relevance to the treatment of infants.

Bacteria↗

A retrospective analysis of the stability and relapse of soft and hard tissue change after bilateral sagittal split osteotomy for mandibular setback of 64 Taiwanese patients.

PURPOSE: This study was an analysis of the soft and hard tissue changes of the facial profile after bilateral sagittal splitting osteotomy for mandibular setback of Taiwanese patients. PATIENTS AND METHODS: We collected pre- and postsurgical lateral cephalographs of 64 patients (28 males, 36 females) with skeletal Class III malocclusion who received combined orthodontic-surgical treatment with bilateral sagittal splitting osteotomy mandibular setback at Taipei Veterans General Hospital between 1994 and 2000. Nineteen cephalometric parameters of (14 linear, 4 angular, and the BS index) soft and hard tissues were measured at 1 week before treatment, and 2 months and 1 year after surgery, and analyzed by paired t test. RESULTS: Mean patient age was 20.0 +/- 1.6 years. The patients underwent an average of 7 mm mandibular setback at the osseous pogonion (Pog). Average setbacks at Pog and soft tissue pogonion (pog) were 5.54 mm and 4.85 mm, respectively, at 1 year after surgery. The setback ratio of Pog/pog was 1:0.88. The hard tissue relapse at Pog was 21% at 1 year after surgery. Improvement in prognathic profile was demonstrated by significant changes in the positions of Pog and pog, ANB angle, the distance from lower lip to esthetic line (E-L lip), and the BS index after surgery. However, compared with parameters obtained from a normal Taiwanese population, the cephalometric data of Pog, pog, and BS index still indicated mild prognathism. CONCLUSION: Although mandibular prognathism could be grossly improved by bilateral sagittal splitting osteotomy mandibular setback, a significant amount of relapse occurred within 1 year after surgery. The extent of the postoperatively preserved features showing mandibular prognathism should be a concern for both patients and physicians.

Adult↗

N-way sensitivity analysis: a verification and stability-assessment technique for completely subjective decision analysis trees.

The purpose of this paper is to describe the development of N-way sensitivity analysis, a modified version of traditional sensitivity analysis that was created for the purpose of verifying the stability of decisions made by a completely subjective decision-analytic tree. The technique was developed during research that addressed whether nurses make clinical decisions that coincide with those recommended by a decision-analytic model. Since all parameters of the model were derived from subjective assessment, traditional one-way or two-way sensitivity analysis was deemed inappropriate. Consequently, N-way sensitivity analysis was developed and used for the verification of the decision model's results.

Analysis of Variance↗

Stability of cisplatin and its monohydrated complex in blood, plasma and ultrafiltrate--implications for quantitative analysis.

The stability of cisplatin and its monohydrated complex has been studied in blood, plasma and plasma ultrafiltrate at 37 degrees C (pH 7.4). Intact cisplatin and the monohydrated complex were determined by liquid chromatography with post-column derivatization. The half lives for cisplatin and the monohydrated complex were 1.43 +/- 0.03 h (SEM) and 0.36 +/- 0.03 h (SEM), respectively, in blood and 0.88 +/- 0.05 h (SEM) and 0.26 +/- 0.02 h (SEM), respectively, in plasma. The compounds were unstable at -25 degrees C (t1/2 for cisplatin was 52 +/- 5 h (SEM) and for the monohydrated compound 26 +/- 2 h (SEM)), but at -70 degrees C both compounds were stable for at least 3 weeks. The monohydrated complex was found to be formed to a small extent when cisplatin was added to plasma (37 degrees C, pH 7.4). A sampling procedure using centripetal ultrafiltration of whole blood was evaluated and found applicable if the samples were stored at 0 degree C and ultracentrifuged within 1 h.

Cisplatin↗

Thermal stability and CD analysis of rat tyrosine hydroxylase.

Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis. It is a homotetramer made up of 56 kDa subunits. We examined the thermal stability of tyrosine hydroxylase purified from a rat pheochromocytoma cell line and investigated the relationship between enzyme activity and stability. Thermal stability was assessed by incubating the enzyme at an elevated temperature. Unfolding of the protein was followed by measuring the loss of circular dichroism (CD) at 220 nm. The CD loss was biphasic, with half-lives of 2 and 14 min at 55 degrees C in 100 mM potassium phosphate, pH 6.0. The rate of loss of enzyme activity paralleled the longer half-life under these conditions. This indicates that the structure of the active site is not appreciably change by the unfolding events corresponding to the first phase. Moreover, unfolding as assessed by the CD spectrum and activity was not reversible and did not exhibit a well-defined midpoint temperature or Tm. The thermal stability of the enzyme was altered by several factors that influence activity. The enzyme at pH 6.0 was less stable (t1/2 = 6.2 and 29 min) than the enzyme at pH 7.2 (a single t1/2 of 64 min). Phosphorylated tyrosine hydroxylase had shorter half-lives (t1/2 of 2 and 16 min) than the nonphosphorylated enzyme (t1/2 6.2 and 29 min) at pH 6.0, 50 degrees C, in 100 mM phosphate. Moderate changes in phosphate concentration had dramatic effects on enzyme stability. Decreasing the phosphate concentration from 50 to 10 mM (pH 6.0) increased the half-life from 2 and 23 min to greater than 120 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Stability testing of solid drugs using preferentially thin-layer chromatography and differential thermal analysis. Part 7: The stability of drugs and preparations (author's transl)].

Beside the temperature etc., certain properties of the crystal surface, the water content, catalytic actions and possible oxidation processes determine the stability of solids. Stability testing of drugs (aiming at fixing time-limits for the storage of drugs) according to the schedule elaborated by the authors provides specific quantitative methods of analysis only for substances of relatively poor storage stability. Already two or three temperature-time experiments (12h/140 degrees C; 20 d/90 degrees C; 43 d/80 degrees C) permit to evaluate the stability of the drugs by thin-layer chromatography and differential thermal analysis. Almost 80% of the substances tested comply with the prerequisites for the recommendation of a time-limit of 10 years for storage.

Catalysis↗

Validation of a computer software program for statistical analysis of accelerated stability studies on biological standards.

Long-term stability is an essential requirement for biological measurement standards and it has been evaluated by applying the Arrhenius model to the data obtained from accelerated thermostability studies. A computer program DEGTEST suited to a mainframe computer has been used for evaluating the stability of biological standards for more than a decade. This paper describes the validation of a computer program executable in a personal computer Microsoft Windows XP environment for the analysis of accelerated thermostability study data.

Biological Products↗

Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis.

Xylose isomerases (XIs) from Thermoanaerobacterium thermosulfurigenes (TTXI) and Thermotoga neapolitana (TNXI) are 70.4% identical in their amino acid sequences and have a nearly superimposable crystal structure. Nonetheless, TNXI is much more thermostable than TTXI. Except for a few additional prolines and fewer Asn and Gln residues in TNXI, no other obvious differences in the enzyme structures can explain the differences in their stabilities. TNXI has two additional prolines in the Phe59 loop (Pro58 and Pro62). Mutations Gln58Pro, Ala62Pro and Gln58Pro/Ala62Pro in TTXI and their reverse counterpart mutations in TNXI were constructed by site-directed mutagenesis. Surprisingly, only the Gln58Pro mutation stabilized TTXI. The Ala62Pro and Gln58Pro/Ala62Pro mutations both dramatically destabilized TTXI. Analysis of the three-dimensional (3D) structures of TTXI and its Ala62Pro mutant derivative showed a close van der Waal's contact between Pro62-C(delta) and atom Lys61-C(beta) (2.92 A) thus destabilizing TTXI. All the reverse counterpart mutations destabilized TNXI thus confirming that these two prolines play important roles in TNXI's thermostability. TTXI's active site has been previously engineered to improve its catalytic efficiency toward glucose and increase its thermostability. The same mutations were introduced into TNXI, and similar trends were observed, but to different extents. Val185Thr mutation in TNXI is the most efficient mutant derivative with a 3.1-fold increase in its catalytic efficiency toward glucose. With a maximal activity at 97 degrees C of 45.4 U/mg on glucose, this TNXI mutant derivative is the most active type II XI ever reported. This 'true' glucose isomerase engineered from a native xylose isomerase has now comparable kinetic properties on glucose and xylose.

Aldose-Ketose Isomerases↗

Rapid analysis of metabolic stability of dopamine receptor antagonists and detection of their metabolites by liquid chromatography/tandem mass spectrometry.

In vitro metabolic stability of dopamine D(3)/D(4) receptor antagonists and identification of their metabolites by high-performance liquid chromatography (HPLC) coupled with ion-trap mass spectrometry (ITMS) were assessed in rat liver microsomes. The compounds were divided into three cassette groups for rapid quantitative analysis of multiple drugs and simultaneous detection of their metabolites. The samples from incubation with rat liver microsomes were pooled into designed cassette groups and analyzed by HPLC/electrospray ITMS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and for 30min. The metabolic stability of the examined dopamine receptor antagonists was in the range of 9.9-84.4%. In addition, the present cassette analysis allowed the simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multistage tandem mass spectrometry on the ion-trap instrument allowed characterization of the structure of the detected metabolites. N,O-dealkylation and ring hydroxylation reactions were identified as major metabolic reactions in piperazinylalkylisoxazole derivatives. These results suggested that the present approach is useful for the rapid evaluation of metabolic stability and structural characterization of metabolites within a short period in new drug discovery.

Alkylation↗

Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein.

Measuring the stability of integrated membrane proteins under equilibrium conditions is hampered by the nature of the proteins' amphiphilic environment. While intrinsic fluorescence is a useful probe for structural changes in water-soluble proteins, the fluorescence of membrane proteins is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a kinetic analysis of the folding of a membrane protein, disulfide bond reducing protein B (DsbB), in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate (SDS) and dodecyl maltoside (DM). This analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. Refolding and unfolding occur on the second to millisecond timescale and involve only one relaxation phase, when monitored by conventional stopped-flow. The kinetic data indicate that denaturation occurs around 0.3 mole fraction of SDS, in agreement with CD analysis and acrylamide quenching data. The rate constants have been fit to a three-state folding scheme involving the SDS-denatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of SDS. The stability of DsbB is around 4.4 kcal/mol in DM, and this is halved upon reduction of the two periplasmic disulfide bonds, and is sensitive to mutagenesis. With the caveat that kinetic data are always open to alternative interpretations, time-resolved studies in mixed micelles provide a useful approach to measure membrane protein stability over a wide range of concentrations of SDS and DM, as well as a framework for the future characterization of the DsbB folding mechanism.

Bacterial Proteins↗

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases.

Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cereus. Several new techniques have been developed to improve the model-building procedures. Also a 'model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophilus. After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress. Hopefully, this approach can be used to unravel the entire early phase of unfolding.

Amino Acid Sequence↗

Stabilization and HPLC analysis of betamethasone sodium phosphate in plasma.

The analysis of corticosteroid prodrugs in pharmacokinetic (PK) studies poses the risk of overestimation of corticosteroid concentrations due to in vitro hydrolysis of prodrugs after sample collection. This study tests the effectiveness of enzyme inhibitors as stabilizers for betamethasone sodium phosphate (BSP) in pregnant sheep plasma samples collected during PK studies with betamethasone (BET) and provides simultaneous high-performance liquid chromatography analysis of BSP and BET. A rapid, sensitive, and specific ion-paired reversed-phase high-performance liquid chromatography assay for simultaneous measurement of BET and BSP in plasma was developed. This assay was used for analyzing samples from an in vitro prodrug hydrolysis study. Enzyme inhibitors tested were sodium arsenate (Na(2)HAsO(4)) and ethylenediaminetetraacetic acid. The BSP was administered intramuscularly to three pregnant sheep to assess in vivo PK. Samples were split with part treated with Na(2)HAsO(4) and part left natural. In vitro hydrolysis of BSP in plasma to BET could be completely inhibited by Na(2)HAsO(4), but not by ethylenediaminetetraacetic acid. The PK study showed lower concentrations of BET in samples with Na(2)HAsO(4) compared with natural samples. This study demonstrates that artifacts in PK profiles of corticosteroids due to in vitro prodrug hydrolysis can be prevented by sample treatment with enzyme inhibitors.

Animals↗

Thermodynamic conformational analysis and structural stability of the nicotinic analgesic ABT-594.

This work presents a theoretical study of the nicotinic analgesic ABT-594. We describe its neutral (precursor) and protonated (active) forms in vacuum and aqueous solution at the MP2/cc-pVDZ level. A conformational analysis is performed on the two torsional angles describing the orientation of the azetidinyl group and the azetidinylmethoxy moiety. To account for entropic effects, a thermostatistical study of conformational populations at physiological temperature is carried out. In the neutral form, conformer I is found as the most populated in vacuum and solution. Here, the nitrogen of the azetidinyl group is far from the electron pairs of the oxygen and the pyridinic nitrogen. In the protonated form, conformer VIII is the most stable in vacuum and solution. Now, the additional proton on the azetidinyl group is oriented toward the electron lone pairs of oxygen. The structural stability of conformers I and VIII is considered through the atoms in molecules theory. The conformer I, in the neutral forms, is stabilized by an intramolecular hydrogen bond. The preference of conformer VIII in the protonated forms is explained by the higher strength of its intramolecular hydrogen bond over the cation-pi interaction found in conformer I. The effect of the interaction energy with the receptor on the conformational preferences of protonated ABT-594 is simulated. The result is that the population of conformers associated to the rotation of the azetidinyl group increases. So, the molecule can easily adopt the optimal internitrogen separation for interaction with the receptor.

Analgesics↗

Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis.

The low-copy-number, 9.0-kb pSM19035-derived plasmid pBT233, is stably inherited in Bacillus subtilis. The complete nucleotide (nt) sequence of pBT233 has been determined. Analysis of the nt sequence revealed nine major open reading frames (orfs). The repS, erm1 and erm2 genes have been assigned to three of these orfs, and given the gene order, repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1. The organization of genes of the repS-orf gamma region resembles the organization of genes in the repE-orfI region of pAM beta 1. Messenger RNA species of molecular weights corresponding to repS, orf alpha + orf beta, orf gamma, orf delta and orf epsilon + orf zeta were detected by Northern blotting. Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 kDa correspond to Orfs beta, gamma, delta, epsilon and zeta, respectively. Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were detected using the T7 promoter-expression system. The orf beta and orf gamma encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively. The orfs, delta, epsilon and zeta, encode proteins with unknown activity. Deletion of a 1.5-kb segment (nt 2999-4552) with coding capacity for orf beta, orf gamma and orf delta does not seem to affect plasmid maintenance. Removal of a 3.0-kb fragment (nt 4598-7689) with coding capacity for orf epsilon and orf zeta reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546-7826) abolished it.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

The role of the coronoid process in elbow stability. A biomechanical analysis of axial loading.

BACKGROUND: The current treatment of coronoid process fractures of the ulna is based on the classification system of Regan and Morrey. We found no biomechanical studies that specifically addressed the role of the coronoid process in elbow stability. In the present investigation, the elbows of cadavera were tested before and after fracture of the coronoid process to assess the stabilizing contribution of the coronoid process under axial loading. METHODS: Six fresh-frozen cadaveric elbows were tested mechanically. All soft tissue surrounding the elbow, including the skin, was left intact. An axial load compressing the elbow joint was applied along the shaft of the forearm in the sagittal plane. A displacement of fifteen millimeters per minute was applied until a load of 100 newtons was attained. Each elbow was tested in 15, 30, 45, 60, 75, 90, 105, and 120 degrees of flexion. Next, less than 25 percent, 25 to 50 percent, or more than 50 percent of the coronoid process was fractured with an osteotome under radiographic guidance, and the testing was repeated. Each elbow served as its own control, and one elbow was used for two tests; therefore, a total of seven situations were investigated. The difference in displacements between the intact and osteotomized elbows was measured. RESULTS: There was no significant difference, at any flexion position, in posterior axial displacement between the intact elbows and the elbows in which 50 percent or less of the coronoid process was fractured (type I and type II) (p = 0.43). There were significant differences, across all flexion positions, in posterior axial displacement between the intact elbows and the elbows in which more than 50 percent of the coronoid process was fractured (type III) (p = 0.006). Specimens with a type-III fracture also showed a significant increase in displacement compared with specimens with a type-I or type-II fracture (p = 0.012). Specifically, from 60 to 105 degrees of flexion, a significant increase in posterior translation of up to 2.4 millimeters was found (p<0.05). CONCLUSIONS: In response to axial load, elbows with a fracture involving more than 50 percent of the coronoid process displace more readily than elbows with a fracture involving 50 percent or less of the coronoid process, especially when the elbow is flexed 60 degrees and beyond.

Adult↗