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High stability and high efficiency chemiluminescent acridinium compounds obtained from 9-acridine carboxylic esters of hydroxamic and sulphohydroxamic acids.

A series of hydroxamic acids and sulphohydroxamic acids were prepared and linked to 9-acridinecarboxylic acid through a pseudo-ester function. After N-methylation of the heterocyclic ring, the different compounds were tested for their chemiluminescent properties. Substituents on the hydroxamic functions have shown various effects (steric or electronic) on the luminescence yield or stability of the molecule. The most interesting derivatives were selected in terms of chemical stability and chemiluminescence efficiency. 9-[(N-hydroxysuccinimidyl-4-oxo-4-N-phenylaminobutanoate)N-carb oxylat e]-10-methyl-acridinium (FA6), 9-(N-phenylpivalamide-N-carboxylate)-10-methylacridinium (FA17) and 9-(N-phenylpivalamide N-carboxylate)-10-carboxymethyl-acridinium (FA18) iodomercurates are very promising as chemiluminescent labels. These compounds can be detected at very low levels (10(-16)-10(-17) mol/L) and in our stability evaluation, FA6, FA17 and FA18 showed similar results to the acridinium ester DMAE. Their half-lives at 20 degrees C are greater than 2 weeks.

Acridines↗

Temporal stability of the implicit association test-anxiety.

The Implicit Association Test-Anxiety (IAT-Anxiety; Egloff & Schmukle, 2002) provides an indirect assessment of anxiety by measuring associations of self (vs. other) with anxiety-related (vs. calmness-related) words. In 3 studies (using 3 independent samples), we examined the temporal stability of the IAT-Anxiety. In Study 1, 65 participants responded twice to the IAT-Anxiety with a time lag of 1 week. The test-retest correlation was .58. In Study 2 (N = 39), we extended the time interval between test and retest to 1 month and this yielded a stability coefficient of .62. In Study 3 (N = 36), we examined the long-term stability (time lag: 1 year) of the IAT-Anxiety and this showed a correlation of .47. We discuss implications of these results for the assessment of personality dispositions with the IAT.

Adult↗

Engineering-enhanced protein secretory expression in yeast with application to insulin.

Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue (Asp(B28)) expressed in yeast was enhanced 5-fold by engineering a specific interaction between an aromatic amino acid in the connecting peptide and a phenol binding site in the hydrophobic core of the molecule. This insulin precursor is characterized by significantly enhanced folding stability. The improved folding properties enhanced the secretion efficiency of the insulin precursor from 10 to 50%. The precursor remains fully in vitro convertible to mature fast-acting insulin.

Insulin↗

Steroid hormones regulate gene expression posttranscriptionally by altering the stabilities of messenger RNAs.

Hormones exert powerful effects on reproductive physiology by regulating gene expression. Recent discoveries in hormone action emphasize that regulation of gene expression is not restricted to their alterations of the rate of gene transcription. On the contrary, hormonal effects on the stability of a specific mRNA can profoundly alter its steady-state concentration. The mRNAs encoding hormone receptors are commonly regulated by their own hormones to create autoregulatory feedback loops. Negative and positive autoregulatory feedback loops serve to limit or augment hormonal responses, respectively. After introducing the topics of mRNA degradation and regulated stability, this review focuses on steroid hormone effects on mRNA stabilities. Autoregulation of the mRNAs encoding estrogen, progesterone, androgen, and glucocorticoid receptors by the steroid hormones in reproductive tissues is discussed. In addition, steroid hormone effects on the stabilities of many other mRNAs that are important to reproductive biology are reviewed. These include mRNAs that encode gonadotropin hormones, integrins, growth factors, and inflammatory response proteins. Through these posttranscriptional effects, steroid hormones impact the expression of a large population of genes. Studies of the molecular mechanisms of hormonally regulated mRNA stabilities continue to identify critical mRNA sequence elements and their interactions with proteins. Increased understanding of how hormones affect mRNA stability may yield novel approaches to the therapeutic control of hormone effects, including those essential to reproductive physiology in animals.

Animals↗

Isolation of Vacuoles from Root Storage Tissue of Beta vulgaris L.

Morphologically intact and osmotically active vacuoles were isolated from root storage tissue of the red beet Beta vulgaris L., and the factors influencing both yield and stability of the vacuoles were determined. Successful isolation depended upon slicing the tissue in an apparatus specifically designed to cut open plant cells without the use of high shear forces and to liberate cellular organelles into an undisturbed reservoir of osmoticum. The resulting brei was centrifuged at 2,000g for 10 min to yield a pellet which contained many vacuoles but which also contained tissue fragments, nuclei, mitochondria, and plastids. The vacuoles were further purified by accelerated flotation through a Metrizamide step gradient. Biochemical assays, light microscopy, and electron microscopy confirmed that there was only trace contamination of the final vacuole preparation by other organelles. Isolated vacuoles were intact and retained their in vivo coloration.

Journal Article↗

Sensory contributions to the control of stance: a posture control model.

We present the outline of a dual kinetic-kinematic postural control model. It is based on concepts of inter-sensory interaction (sensor fusion) which we consider instrumental for sensorimotor integration. Separation into kinetic and kinematic control signals begins at the level of the sensors (e.g., vestibular system--otoliths: force field meters, canals: head angular speedometers). Sensor fusion mechanisms are used to yield separate internal representations for foot support kinematics, force fields such as gravity, and contact forces such as pull or push having impact on the body. These representations are fed as global set point signals into local proprioceptive control loops of the joints. Fed into an ankle joint proprioceptive loop for body-on-support stabilization, they yield compensation of support tilt, gravity and contact forces, even when these stimuli are combined and, furthermore, voluntary lean is superimposed. Model simulations parallel our experimental findings so far obtained.

Animals↗

Contribution of central and peripheral vision to the regulation of stance: developmental aspects.

Postural oscillations in 6-, 8-, and 10-year-old children were analyzed in four conditions of vision of the environment (complete vision, peripheral vision, central vision and no-vision) and two conditions of ankle somatosensory information (normal and altered support surfaces with a 5-cm-thick foam). Children were more stable with than without vision. This was observed whether children had complete or partial vision (central or peripheral). They were also more stable with the normal than with the altered support surface. Overall, there was no effect of age. Beyond these well-established results, the present experiment showed the complementary role of peripheral and central vision in the regulation of children's posture. For the 6- and 10-year-olds, central and peripheral vision yielded similar postural stability, whereas for the 8-year-olds, central vision yielded greater postural stability than peripheral vision. The analysis of postural oscillations in the medio-lateral and antero-posterior planes showed that, for the three age groups, central vision was as efficient whatever the plane. On the other hand, after age 6, peripheral vision was more efficient for regulating antero-posterior than medio-lateral oscillations. The contribution of the different sensory systems and their interaction for stabilizing posture in children should be specifically interpreted with regard to the operating characteristics of each sensory system at each age.

Age Factors↗

Base pairing involving deoxyinosine: implications for probe design.

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.

Hydrogen Bonding↗

Plant breeding and drought in C3 cereals: what should we breed for?

Drought is the main abiotic constraint on cereal yield. Analysing physiological determinants of yield responses to water may help in breeding for higher yield and stability under drought conditions. The traits to select (either for stress escape, avoidance or tolerance) and the framework where breeding for drought stress is addressed will depend on the level and timing of stress in the targeted area. If the stress is severe, breeding under stress-free conditions may be unsuccessful and traits that confer survival may become a priority. However, selecting for yield itself under stress-alleviated conditions appears to produce superior cultivars, not only for optimum environments, but also for those characterized by frequent mild and moderate stress conditions. This implies that broad avoidance/tolerance to mild-moderate stresses is given by constitutive traits also expressed under stress-free conditions. In this paper, we focus on physiological traits that contribute to improved productivity under mild-moderate drought. Increased crop performance may be achieved through improvements in water use, water-use efficiency and harvest index. The first factor is relevant when soil water remains available at maturity or when deep-rooted genotypes access water in the soil profile that is not normally available; the two latter conditions become more important when all available water is exhausted by the end of the crop cycle. Independent of the mechanism operating, a canopy able to use more water than another would have more open stomata and therefore higher canopy temperature depression, and 13C discrimination (delta13C) in plant matter. The same traits would also seem to be relevant when breeding for hot, irrigated environments. Where additional water is not available to the crop, higher water-use efficiency (WUE) appears to be an alternative strategy to improve crop performance. In this context delta13C constitutes a simple but reliable measure of WUE. However, in contrast to lines performing better because of increased access to water, lines producing greater biomass due to superior WUE will have lower delta13C values. WUE may be modified not only through a decrease in stomatal conductance, but also through an increase in photosynthetic capacity. Harvest index is strongly reduced by terminal drought (i.e. drought during grain filling). Thus, phenological traits increasing the relative amount of water used during grain filling, or adjusting the crop cycle to the seasonal pattern of rainfall may be useful. Augmenting the contribution of carbohydrate reserves accumulated during vegetative growth to grain filling may also be worthwhile in harsh environmcnts. Alternatively, extending the duration of stem elongation without changing the timing of anthesis would increase the number of grains per spike and the harvest index without changing the amount of water utilized by the crop.

Acclimatization↗

Enzyme reaction engineering: design of peptide synthesis by stabilized trypsin.

By using very active and very stable trypsin agarose derivatives, we have optimized the design of the synthesis of a model dipeptide, benzoylarginine leucinamide, by two different strategies: (i) kinetically controlled synthesis (KCS), by using benzoyl arginine ethyl ester and leucinamide as substrates, and (ii) thermodynamically controlled synthesis (TCS), by using benzoyl arginine and leucinamide as substrates. In each strategy, we have studied the integrated effect of a number of variables that define the reaction medium on different parameters of industrial interest, e.g. time course of peptide synthesis, higher synthetic yields, and stability of the catalyst, as well as aminolysis/hydrolysis ratios and rate of peptide hydrolysis in the case of KCS. Both synthetic approaches were carried out in monophasic water or water-organic cosolvent systems. We have mainly tested a number of variables, e.g. temperature, polarity of the reaction medium (presence of cosolvents, presence of ammonium sulfate), and exact structure of the trypsin derivatives. Optimal experimental conditions for these synthetic approaches were established in order to simultaneously obtain good values for all industrial parameters. The use of previously stabilized trypsin derivatives greatly improves the design of these synthetic approaches (e.g. by using drastic experimental conditions: 1 M ammonium sulfate (KCS) or 90% organic cosolvents (TCS]. In these conditions, our derivatives preserve more than 95% of activity after 2 months and we have been able to reach synthetic productivities of 180 (KCS) and 1 (TCS) tons of dipeptide per year per liter of catalyst.

Dipeptides↗

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue.

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6-8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (less than 5 micrograms/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent Km of 60 microM and Vmax of 20 mumol X min-1 X mg-1, corresponding values with mixed histones were 12 microM and 1.2 mumol X min-1 X mg-1. With both protein substrates the apparent Km for ATP was 11 microM. Concentrations of Mg2+ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7-2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated 32Pi from [gamma-32P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.

Adipose Tissue↗

Generation of microtubule stability subclasses by microtubule-associated proteins: implications for the microtubule "dynamic instability" model.

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.

Animals↗

Crop responses to climatic variation.

The yield and quality of food crops is central to the well being of humans and is directly affected by climate and weather. Initial studies of climate change on crops focussed on effects of increased carbon dioxide (CO2) level and/or global mean temperature and/or rainfall and nutrition on crop production. However, crops can respond nonlinearly to changes in their growing conditions, exhibit threshold responses and are subject to combinations of stress factors that affect their growth, development and yield. Thus, climate variability and changes in the frequency of extreme events are important for yield, its stability and quality. In this context, threshold temperatures for crop processes are found not to differ greatly for different crops and are important to define for the major food crops, to assist climate modellers predict the occurrence of crop critical temperatures and their temporal resolution. This paper demonstrates the impacts of climate variability for crop production in a number of crops. Increasing temperature and precipitation variability increases the risks to yield, as shown via computer simulation and experimental studies. The issue of food quality has not been given sufficient importance when assessing the impact of climate change for food and this is addressed. Using simulation models of wheat, the concentration of grain protein is shown to respond to changes in the mean and variability of temperature and precipitation events. The paper concludes with discussion of adaptation possibilities for crops in response to drought and argues that characters that enable better exploration of the soil and slower leaf canopy expansion could lead to crop higher transpiration efficiency.

Acclimatization↗

Use of polypropylene mesh to stabilize the "floating humerus" after total scapulectomy.

Total scapulectomy for malignant disease is a rarely performed procedure that presents a significant challenge for shoulder reconstruction. Failure to stabilize the resulting "floating humerus" may result in significant esthetic and functional problems. Current techniques of reconstruction and stabilization may yield suboptimal results and significant morbidity. We report a case of Ewing's sarcoma of the scapula, which required total scapulectomy. Polypropylene mesh was used in an attempt to prevent migration of the head of the humerus. The result was a stable shoulder with satisfactory motion and no additional morbidity. We believe that polypropylene mesh offers an advantage in shoulder reconstruction after total scapulectomy and should be considered as an option for stabilization of the humerus.

Adult↗

Energy dissipation and stability of propagating surfaces.

Thermodynamic equilibrium states are given by the minimum of a convex free energy function with suitable boundary conditions. Nonconvexity may lead to the coexistence of several phases and the classical Gibbs phase rule allows constructing their equilibrium properties (e.g., density or pressure). Within the framework of nonequilibrium thermodynamics, the maximization of energy dissipation (under suitable boundary conditions) can be used as an extremal principle to find stationary states. We show that stationary states generally exist for convex energy dissipation functions and that nonconvexity leads to metastable and unstable states. A geometric argument, similar in spirit to Gibbs' double-tangent construction, yields the stability limits of stationary states. This argument is applied to study a classical problem of materials science, namely the motion of a grain boundary under the influence of solute drag.

Journal Article↗

Temporal stability of the emotion-modulated startle response.

In the present study, we examined the stability of one measure of emotion, the emotion-modulated acoustic startle response, in an undergraduate sample. Using the acoustic startle paradigm on two different occasions, we measured stability of affective modulation of the startle response during and following the presentation of pictures selected to be of positive, negative, or neutral emotional valence. The two assessments were separated by 4 weeks. Two groups of subjects were compared: one group that viewed the same pictures at each assessment and a second group that viewed different pictures at the second assessment. We found that viewing different pictures at two assessments separated by 4 weeks yielded moderate stability of the emotion modulation of startle magnitude, whereas subjects who viewed the same pictures at both assessments showed poor stability. Furthermore, this difference was due to the stability of responses to high versus low arousal pictures, not to differences in valence.

Adolescent↗

Characterization of chemically crosslinked human factor VIIIa.

The activity of factor VIIIa was enhanced and stabilized by treatment of factor VIII with the crosslinker disuccinimidyl suberate. The activity was > 200-fold higher compared with that of native factor VIIIa and was stable for at least 15 days at 4 degrees C and pH 7.2. The crosslinked factor VIIIa was purified by immunoaffinity chromatography and gel filtration. Electrophoretic analysis revealed high-molecular-mass (approximately 150 kDa) molecules as well as the three bands characteristic of native factor VIIIa. Thus crosslinking appeared to yield molecules stabilized by intra- and/or inter-subunit crosslinks. The material was further fractionated using immobilized von Willebrand factor and the factor VIIIa activity could be ascribed to trimers containing only intra-subunit crosslinks. Moreover, reduction of crosslinked factor VIIIa produced using dithiobis(succinimidylpropionate) suggested that the molecules containing intersubunit crosslinks had not been cleaved by thrombin at arginine 372.

Chromatography, Affinity↗

Formulation and stability of freeze-dried proteins: effects of moisture and oxygen on the stability of freeze-dried formulations of human growth hormone.

This research presents the results of a series of stability studies on freeze-dried formulations of human growth hormone (hGH). Chemical decomposition via methionine oxidation and asparagine deamidation as well as irreversible aggregation are characterized by HPLC. Water sorption isotherms, DSC thermograms, and pulsed proton NMR data are also obtained. No glass transition temperatures are observed in the temperature range of the stability studies. The pulsed NMR data suggest onset of greater mobility in the solid at a water content slightly higher than BET "monolayer" level. Stability of freeze-dried solids at 25 degrees C and 40 degrees C is studied as a function of residual moisture and exposure to oxygen. Formulations with and without a glycine/mannitol excipient system are studied. Significant levels of chemical decomposition and irreversible aggregation occur under most conditions with the effects of residual water content and "headspace oxygen" strongly dependent on the formulation. At low water content with minimal oxygen in the vial headspace, the glycine/mannitol formulation yields optimum stability. However, for either high water content or high oxygen content in the vial, stability of hGH without excipients is superior. The qualitative effect of residual moisture on stability depends on the temperature of the stability study. Generally, the stability of a sample adjusted to a given water content by desorption (during freeze-drying) is identical to the stability of a sample prepared by sorption of water on to a previously highly dried sample.

Calorimetry, Differential Scanning↗