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PubMed2006

Calorimetric analysis of thermodynamic stability and aggregation for apo and holo amyotrophic lateral sclerosis-associated Gly-93 mutants of superoxide dismutase.

Differential scanning calorimetry was used to measure changes in thermodynamic stability and aggregation for glycine 93 mutants of human copper, zinc-superoxide dismutase (SOD). Glycine 93 is a conserved residue at position i + 3 of a tight turn and has been found to be a mutational hot spot in familial amyotrophic lateral sclerosis (fALS). The fALS-associated mutations, G93A, G93S, G93R, G93D, and G93V, were made in a pseudo wild-type background containing no free cysteines, which prevented the formation of aberrant disulfide bonds upon thermal unfolding, and enabled quantitative thermodynamic analysis of the effects of the mutations. Thermal unfolding was highly reversible for all the SODs in both the fully metallated (holo) and metal-free (apo) forms. The data for all the holo-SODs and for the apo-pseudo-wild-type SOD were well fit by a 2-state unfolding model for native dimer (N2) to two unfolded monomers (2U), N2 <--> 2U. The holo- and apo-forms of the mutants are significantly destabilized (by 1.5-3.5 kcal mol(-1) monomer) relative to the corresponding forms of pseudo wild-type, with the relative stabilities being correlated with statistical preferences for amino acids in this structural context. Although van't Hoff (DeltaHvH) to calorimetric (DeltaHcal) enthalpy ratios are close to unity for all the holo-SODs and for apo-pseudo-wild-type, consistent with a 2-state transition, DeltaHvH is considerably larger than DeltaHcal for all the apo-mutants. This suggests that the mutations cause apo-SOD to have an increased propensity to misfold or aggregate, which may be linked to increased toxic mutant SOD aggregation in fALS.

Amino Acid Substitution↗
PubMed2000

Stability and haplotype analysis of the FRAXE region.

FRAXE full mutations are rare and appear to be associated with mild mental retardation. As part of a screening survey of boys with learning difficulties to determine the frequency of full and premutations, we have collected data on the frequency of instability at FRAXE for about 4000 transmissions and the haplotype for over 7000 chromosomes. The distribution of FRAXE repeats was similar to other English populations but differed from two North American Caucasian series. Observed instability at FRAXE was rare but increased with increasing repeat number, and there were no expansions into the full mutation range, except in pedigrees ascertained through a full mutation. Haplotype analysis suggested division into five groups with each group having a characteristic distribution of FRAXE repeats. Fourteen of the 15 full mutations occurred on a single haplotype and this haplotype also had a significant excess of intermediate-sized alleles, suggesting that full mutations originate from large normal alleles. However, a related haplotype also had a significant excess of intermediates but we observed no full mutations on this haplotype, suggesting either loss or gain of stability determinants on it. We suggest that whilst triplet repeat size is a significant predisposing factor for expansion at FRAXE other genetic determinants are also likely to be important.

Alleles↗
PubMed1998

Computational analysis of thermal stability: effect of Ile-->Val mutations in human lysozyme.

BACKGROUND: Free energy calculations are carried out to study the change of thermal stability caused by Ile23-->Val, Ile56-->Val, Ile89-->Val and Ile106-->Val mutations in human lysozyme. In order to examine the dependence of the free energy difference, DeltaDeltaG, on the denatured-state structure, extended and native-like conformations are employed as initial conformations in the denatured-state simulations. RESULTS: Calculated values of DeltaDeltaG for the mutations, Ile56-->Val, Ile89-->Val and Ile106-->Val, were in good agreement with experimental values when the native-like structure was employed in the respective denatured-state simulations. In the case of Ile23-->Val, a considerable difference between the calculated and experimental values of DeltaDeltaG was observed. CONCLUSIONS: The physical nature of Ile56-->Val, Ile89-->Val and Ile106-->Val mutations was rationally characterized by a free energy component analysis. It is suggested that the alpha domain in which Ile23 is included is considerably structured even in the denatured state.

Algorithms↗
PubMed2002

Glass-fiber self-mixing intra-arterial laser Doppler velocimetry: signal stability and feedback analysis.

We have developed a blood velocimeter based on the principle of self-mixing in a semiconductor laser diode through an optical fiber. The intensity of the light is modulated by feedback from moving scattering particles that contain the Doppler-shift frequency. Upon feedback the characteristics of the laser diode change. The threshold current decreases, and an instable region may become present above the new threshold. The amplitude of the Doppler signal turns out to be related to the difference in intensity between situations with and without feedback. This amplitude is highest just above feedback. The suppression of reflection from the glass-fiber facets is of paramount importance in the obtaining of a higher signal-to-noise ratio. Using an optical stabilization of the feedback, we optimized the performance of the laser-fiber system and the Doppler modulation depth and clarified its behavior with a suitable physical model. We also investigated the effect of the finite coherence length of the laser. We tested the efficiency of the self-mixing velocimeter in vivo with the optical glass fiber inserted in the artery with endoscopic catheters, both in upstream and in downstream blood flow conditions. For the latter we used a special side-reflecting device solution for the fiber facet to allow downstream measurements.

Animals↗
PubMed1999

Analysis of synthesis, stability, phosphorylation, and interacting polypeptides of the 34-kilodalton product of open reading frame 6 of the early region 4 protein of human adenovirus type 5.

The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.

Adenovirus E1B Proteins↗
PubMed2004

Cold-temperature stability of five beta-lactam antibiotics in bovine milk and milk extracts prepared for liquid chromatography-electrospray ionization tandem mass spectrometry analysis.

The stability of five major beta-lactam antibiotics (amoxicillin, ampicillin, cloxacillin, oxacillin, and penicillin G) in fortified milk and in milk extracts prepared for LC-ESIMS/MS analysis was studied at varying cold temperatures (4, -20, and -76 degrees C). Storage of milk samples at 4 degrees C resulted in measurable losses of all beta-lactams after 6 days (>50% in most cases). Slow degradation of penicillin G, cloxacillin, and oxacillin was observed in milk stored at -20 degrees C, but no losses were recorded at -76 degrees C over 4 weeks. All antibiotics showed good stability at all temperature tested in milk extracts prepared for LC-ESIMS/MS analysis. The results of this study emphasize adherence to adequate sample handling and storage protocols as to reflect residue levels at the time of sample submission.

Animals↗
PubMed1996

A biomechanical analysis of the stability of titanium bone fixation systems in proximal phalangeal fractures.

Apex bending and torsional loading were utilized to study the effects of different plate design and thickness, and screw size and design on the rigidity and strength of seven different titanium mini- and microplates placed onto osteotomized proximal phalanges. One hundred forty-four fresh frozen human cadaveric proximal phalangeal bones underwent a mid shaft osteotomy followed by application of one of the following plates: (1) Synthes linear 1.5-mm five-hole plates, (2) Leibinger linear 1.2-mm five-hole or (3) 1.7-mm four-hole plates, or (4) Leibinger three-dimensional 1.2-mm four-hole, (5) 1.2-mm eight-hole, (6) 1.7-mm four-hole, or (7) 1.7-mm eight-hole plates. Three-point bending (apex dorsal or apex volar) and torsional loading were utilized for each plating configuration. Analysis of variance models of bone specimen width, depth, cortical thickness, and length revealed that increasing plate thickness was associated with increasing rigidity, but that the three-dimensional design yielded a higher relative rigidity except under apex volar loading.

Biomechanical Phenomena↗
PubMed1998

Analysis of the stability of hemoglobin S double strands.

The deoxyhemoglobin S (deoxy-HbS) double strand is the fundamental building block of both the crystals of deoxy-HbS and the physiologically relevant fibers present within sickle cells. To use the atomic-resolution detail of the hemoglobin-hemoglobin interaction known from the crystallography of HbS as a basis for understanding the interactions in the fibers, it is necessary to define precisely the relationship between the straight double strands in the crystal and the twisted, helical double strands in the fibers. The intermolecular contact conferring the stability of the double strand in both crystal and fiber is between the beta6 valine on one HbS molecule and residues near the EF corner of an adjacent molecule. Models for the helical double strands were constructed by a geometric transformation from crystal to fiber that preserves this critical interaction, minimizes distortion, and makes the transformation as smooth as possible. From these models, the energy of association was calculated over the range of all possible helical twists of the double strands and all possible distances of the double strands from the fiber axis. The calculated association energies reflect the fact that the axial interactions decrease as the distance between the double strand and the fiber axis increases, because of the increased length of the helical path taken by the double strand. The lateral interactions between HbS molecules in a double strand change relatively little between the crystal and possible helical double strands. If the twist of the fiber or the distance between the double strand and the fiber axis is too great, the lateral interaction is broken by intermolecular contacts in the region around the beta6 valine. Consequently, the geometry of the beta6 valine interaction and the residues surrounding it severely restricts the possible helical twist, radius, and handedness of helical aggregates constructed from the double strands. The limitations defined by this analysis establish the structural basis for the right-handed twist observed in HbS fibers and demonstrates that for a subunit twist of 8 degrees, the fiber diameter cannot be more than approximately 300 A, consistent with electron microscope observations. The energy of interaction among HbS molecules in a double strand is very slowly varying with helical pitch, explaining the variable pitch observed in HbS fibers. The analysis results in a model for the HbS double strand, for use in the analysis of interactions between double strands and for refinement of models of the HbS fibers against x-ray diffraction data.

Amino Acid Sequence↗
PubMed2026

Grain protein and yield stability study in rainfed durum wheat RILs.

Developing durum wheat cultivars with stable grain yield across diverse environments remains a key breeding objective. This study evaluated 118 recombinant inbred lines (RILs) derived from a cross between the drought-adapted cultivar 'Zardak' (Triticum durum) and the landrace 'Iran-249' (T. turanicum) with desirable seed characteristics, across four heterogeneous rainfed environments in Italy and Iran. The assessment focused on grain yield (GY) and grain protein content (GPC) stability. Combined analysis of variance revealed significant (p&#x2009;<&#x2009;0.01) effects for genotype, environment, and their interaction for both traits. Line ZD-050 showed the highest GY (3.91 t ha&#x207b;&#xb9;), while ZD-032 had the highest GPC (14.27%). Stability analysis using parametric and non-parametric methods, along with AMMI and GGE biplot modeling, identified ZD-050 as among the most promising genotypes according to yield-integrating and dynamic-stability approaches. This line showed high grain yield in methods such as the Superiority Index and Kang's rank-sum, although stability rankings differed across the used methods. This line maintained superior yield, demonstrated broad adaptability across environments, and had moderate protein levels, identifying it as an optimal candidate for breeding programs targeting yield stability and wide adaptation under rainfed conditions.

Triticum↗
PubMed1976

High-sensitivity linear dichroism as a tool for equilibrium analysis in biochemistry. Stability constant of DNA--ethidiumbromide complex.

A stoichiometrical application of a sensitive method for linear dichroism (LD) detection is suggested for biochemical purposes. The complex formation between a binding site on a polynucleotide and a ligand may be studied with high precision if the following conditions are fulfilled: (1) The polymer can be given a fixed degree of orientation. (2) The site has a specific orientation with respect to the orientation axis of the polymer (e.g., intercalation). (3) The ligand has an anisotropic optical absorption property. The method was applied to studying the complex between DNA and ethidiumbromide, which was detected by LD with precision of +/- 0.5 X 10(-7) M in a 4 X 10(-4) M DNA solution, i.e., 0.1% occupation of the total site concentration can be detected. The complexation could be explained by a single type of site (n = 0.14 +/- 0.01 sites per nucleotide residue) and a stability constant K1 = (2.5 +/- 1) X 10(5) M-1 at 0.2 M ionic strength. From the specific LD an average angle 60 degrees was concluded between the helix axis and the long axis of the ethidiumbromide molecule. This value formally contradicts the Watson-Crick model or the intercalation model but may be explained by extension and deformation effects on the xhain by the flow.

Binding Sites↗
PubMed1998

Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames.

Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448-1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (gfp) of Aequorea victoria or the gag gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3Cpro/3CDpro cleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins > 10 kDa in size through fusion with the polyprotein needs to be reevaluated.

Amino Acid Sequence↗
PubMed2003

Analysis of genetic stability at the EP300 and CREBBP loci in a panel of cancer cell lines.

EP300 (p300) and CREBBP (CBP) are highly related transcriptional co-activators possessing histone acetyltransferase activity. These proteins have been implicated in coordinating numerous transcriptional responses that are important in the processes of proliferation and differentiation. A role for EP300 and CREBBP as tumor suppressors in cancer has been suggested by the fact that they are targeted by viral oncogenes; there is an increased incidence of hematologic malignancies in mice monoallelic for CREBBP; and loss, albeit at a low frequency, of both EP300 alleles in epithelial cancers has been observed. Because the level of EP300/CREBBP appears to have a critical effect on integrating certain transcriptional processes, we sought to determine whether a loss in the combined gene dosage of EP300 and CREBBP might play a role in cancer. Accordingly, we screened a panel of 103 cell lines for loss of heterozygosity and found 35 and 51% LOH for the CREBBP and EP300 loci, respectively. Concordant loss of CREBBP and EP300 was not associated with mutations in important regions of the remaining EP300 or CREBBP genes. In addition, there did not appear to be a statistically significant selection in cancer cells, stratified by various criteria, for the concordant loss of EP300 and CREBBP. We conclude that EP300 and CREBBP rarely act as classical tumor suppressors in human cancer.

CREB-Binding Protein↗
PubMed2000

DNA condensation by protamine and arginine-rich peptides: analysis of toroid stability using single DNA molecules.

Both somatic cells and sperm have been shown to take up exogenous DNA, but the frequency of its integration is usually low. Scanning probe microscopy studies of sperm chromatin and synthetic DNA-protamine complexes indicate that the coiling of DNA into toroidal subunits, a process initiated in the maturing spermatid to prepare its genome for delivery into the egg, can be mimicked by simply adding protamine to DNA in vitro. The increased resistance of DNA-protamine complexes to nuclease digestion and their structural similarity to native sperm chromatin suggest that the packaging of DNA by protamine might offer a new approach for improving the efficiency of DNA uptake by sperm. Decondensation experiments performed with individual DNA molecules have provided a direct measure of the stability of toroids produced using salmon protamine and smaller arginine-rich peptides. These experiments show that the arginine content of protamine-related sequences can have a dramatic effect on their rate of dissociation from DNA. This technique and the information it provides can be used to identify protamine analogs that can be bound to DNA to increase the efficiency of its uptake by sperm and other cells.

Arginine↗
PubMed2001

Development of small-size tubular-flow continuous reactors for the analysis of operational stability of enzymes in low-water systems.

A very small-scale continuous flow reactor has been designed for use with enzymes in organic media, particularly for operational stability studies. It is constructed from fairly inexpensive components, and typically uses 5 mg of catalyst and flow rates of 1 to 5 mL/h, so only small quantities of feedstock need to be handled. The design allows control of the thermodynamic water activity of the feed, and works with temperatures up to at least 80 degrees C. The reactor has been operated with both nonpolar (octane) and polar (4-methyl-pentan-2-one) solvents, and with the more viscous solvent-free reactant mixture. It has been applied to studies of the operational stability of lipases from Chromobacterium viscosum (lyophilized powder or polypropylene-adsorbed) and Rhizomucor miehei (Lipozyme) in different experimental conditions. Transesterification of geraniol and ethylcaproate has been adopted as a model transformation.

Acyclic Monoterpenes↗
PubMed1993

FRAP analysis of the stability of the microtubule population along the neurites of chick sensory neurons.

In order to study microtubule turnover in elongating neurites, chick embryo sensory neurons were microinjected with x-rhodamine tubulin, and after 6-12 hours, short segments along chosen neurites were photobleached at multiple sites. Previous studies [Lim et al., 1989; 1990] indicated that recovery of fluorescence (FRAP) in neurites occurs by the dynamic turnover of stationary microtubules. In all cases, distal bleached zones recovered fluorescence faster than bleached zones more proximally located along the same neurites. Bleached zones at growth cones completely recovered in 30-40 minutes, while bleached zones located more proximally usually recovered in 50-120 minutes. In the most proximal regions of long neurites, recovery of fluorescence was often incomplete, indicating that a significant fraction of the microtubules in these regions were very stable. These studies indicate that there are differences in microtubule stability along the length of growing neurites. These differences may arise from the combined effects of 1) modifications that stabilize and lengthen microtubules in maturing neurites and 2) the dynamic instability of the distally oriented microtubule plus ends.

Animals↗
PubMed2007

Solid phase extraction as a faster alternative to HPLC: application to MS analysis of metabolic stability samples.

Due to recent advances in high throughput organic synthesis, discovery teams now need to profile increased numbers of analogs in vitro for their absorption, distribution, metabolism, and excretion (ADME) properties. Consequently, pharmaceutical companies are developing lower cost and higher throughput methods for ADME testing. As demands for metabolic stability testing have increased in our laboratory, the time required to analyze samples using high-pressure liquid chromatography-mass spectrometry (HPLC-MS) has grown rapidly and ultimately limited our data output. In this study we show that solid phase extraction-mass spectrometry (SPE-MS) is a viable alternative to HPLC-MS for monitoring small molecule stability in liver microsomes. Using the SPE-MS approach, samples can be analyzed in 24 s compared to 2.5 min on the HPLC-MS without compromising data quality, thereby alleviating the analytical bottleneck.

Animals↗