Genetical analysis on the stability of poliovirus type 3 Leon 12a1b.
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Poliovirus↗
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Analysis of thermal stability of soya globulins using monoclonal antibodies.
The epitopes of two monoclonal antibodies (Mabs), one raised to soya glycinin (IFRN 0025) and one to beta-conglycinin (IFRN 0089), have been defined. The epitope of 0025 corresponds to residues 86-104 of the acidic polypeptide of glycinin A1aB1b and lies at the C terminus of the proteolytic intermediate known as glycinin-T, whilst that of 0089 corresponds to residues 78-84 in the acidic extension present in the alpha' subunit of beta-conglycinin. As the Mabs bind to synthetic peptides corresponding to the epitope regions both epitopes can be considered as being continuous in nature. The regions recognised correspond to surface loops, which are probably flexible in nature. Both Mabs were used to investigate thermally induced conformational changes in soya globulins. Thermally treated glycinin was recognised more strongly than native protein, possibly due to disruption of the disulphide bond joining the acidic and basic polypeptides. Disruption could increase epitope accessibility, as could the conformational changes associated with denaturation. The binding of anti-beta-conglycinin Mab 0089 was unaffected by heating, suggesting that its epitope remains on the surface of the aggregates formed on heating. This study demonstrates that Mabs with defined specificities can be sensitive probes for monitoring local conformational changes within a protein molecule during thermal denaturation.
Amino Acid Sequence↗
A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase.
The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degrees C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degrees C and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degrees C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degrees C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.
Chemical Phenomena↗
Solidification/stabilization of electric arc furnace dust using coal fly ash. Analysis of the stabilization process.
In this paper, the stabilization of electric arc furnace (EAF) dust containing hazardous metals such as Pb, Cd, Cr or Zn is described. The treatment involves a waste solidification/stabilization (S/S) process, using coal fly ash as the fundamental raw material and main binder. The article also contains a brief review of the most important recent publications related to the use of fly ash as S/S agents. The efficacy of the process has been evaluated mainly through leaching tests on the solidified products and compliance with some imposed leachate limits. The concentration of metals leaching from the S/S products was strongly leachate pH dependent; thus, the final pH of the leachate is the most important variable in reaching the limits and, therefore, in meeting the stabilization goals. In this study, the dependence relationship between the leachate pH and the concentrations of metals in the leachate are analyzed; in some cases, this allows us to estimate the speciation of contaminants in the S/S solids and to understand the mechanism responsible for reduced leachability of heavy metals from solidified wastes.
Coal↗
Small angle neutron scattering analysis of thermal stability of 23S rRNA and the intact 50S subunits of Sulfolobus solfataricus.
The ribosomes of the extremely thermophilic archaebacterium, Sulfolobus solfataricus, are very resistant to thermal denaturation (optimal growth temperature 87 degrees C), remaining essentially intact up to above 90 degrees C. However, the separate ribosomal components (rRNA and r-proteins) are less thermally stable than the ribosome as a whole, indicating that the mode of interaction of all of the components within the ribonucleoprotein particle play an essential role in determining thermal stability. To get some insight into the structural features of the thermophilic ribosome, we performed small angle neutron scattering (SANS) measurements at various temperatures on Sulfolobus solfataricus intact large ribosomal subunits (50S) and deproteinated large ribosomal subunit RNA (23S). Even if the scattering profiles suggest the presence of supramolecular aggregates in all of the samples and at all of the investigated temperatures, the measured form factors indicated for both samples that, at temperatures above 70 degrees C, the suspended particles underwent a structural rearrangement. This finding is likely to reflect single particles' properties, since S. solfataricus ribosomes are known to be biologically activated only above 60 degrees C, and there are indications that such activation requires a conformational rearrangement of the particle. A remarkable superimposition of the percentage variation of the volume from neutron scattering and of the absorbency increment with respect to temperature supports this view.
Neutrons↗
Haliotis tuberculata hemocyanin (HtH): analysis of oligomeric stability of HtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2.
The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31-41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43-56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329-339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100mM concentrations of calcium and magnesium ions in Tris-HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.
Animals↗
Xerox copy of the dental cast for analysis of the stability of mandibular osteotomy.
Stability of the mandible was analysed with a Xerox copy of the dental cast in 35 cases of mandibular prognathism corrected by ramus osteotomies. The occlusal relationship in the Xerox copy was illustrated by superimposing two contact points on the upper and lower dental arches and the change in the position of the lower dental arch in relation to the upper dental arch was obtained by superimposing the latter on preoperative, predicted postoperative, and six months postoperative occlusions. The stability of the mandible was analysed by measuring the movement of five landmarks (two posterior, two molar and one incisor points) set around the lower dental arch to represent the movement of the mandible. The relapse was estimated by the movements of the landmarks from the predicted postoperative occlusion to the six months postoperative occlusion. The mean estimated anterior relapses at the posterior and molar points with the larger predicted movement and the incisor point were between 0.9 mm. and 2.0 mm. at six months postoperatively, whereas on the side with the smaller predicted movement, the points moved posteriorly by 0.6 mm. and 0.5 mm. Despite the fact that the amount of the predicted lateral movement was much smaller than that of the predicted posterior movement at operation, the posterior points were estimated to relapse laterally by 2.0 mm. and 1.7 mm.(ABSTRACT TRUNCATED AT 250 WORDS)
Cephalometry↗
Melting-profile analysis of thermal stability of thermolysin. A formulation of temperature-scanning kinetics.
The melting-profile method consists of a continuous observation of a structural parameter while the temperature of the sample is raised at a constant rate [Fugita, S. C., & Imahori, K. (1974) IN Peptides, Polypeptides and Proteins (Blout, E. R., Bovey, F. A., Goodman, M., & Lotan, N., Eds.) p 217, Wiley, New York, N.Y.]. An analytical solution to the melting profile was formulated for the two-state irreversible process and called temperature-scanning kinetics. The theory was tested with thermolysin with consistent results, and the thermodynamic parameters of thermal denaturation were calculated: deltaH identical to = 80.3 kcal/mol, deltaS identical to = 153 eu. These values agreed with the corresponding values obtained from the classical constant-temperature relaxation kinetics. The possibilities of temperature-scanning kinetics are discussed.
Calcium↗
Clinical grade vector production: analysis of yield, stability, and storage of gmp-produced retroviral vectors for gene therapy.
Retroviral vector gene transfer of a therapeutic gene to correct or modify a disease process is a promising strategy for many inherited and acquired diseases. A major obstacle in this process is the large-scale production of the gene transfer vector under good manufacturing practice (GMP) conditions. We have used the CellCube bioreactor system to produce five batches of GMP-grade vector. The production batches were of 10-20 L each, and the titers were around 2 x 10(6) IU/mL. We find that this particular vector is relatively stable with a half-life of about 8 h at 37 degrees C, 40 h at 20 degrees C, and 14 days at 4 degrees C. The half-life during storage at -80 degrees C is around 18 months. The supernatant may be frozen and thawed up to five times without any significant loss of titer. We have also made a comparison between the CellCube bioreactor and the automated roller bottle system RollerCell 40 (RC 40). The yields from the two systems were comparable.
Base Sequence↗
Quantitative analysis, occurrence, and stability of (E)-1-(2,3,6-Trimethylphenyl)buta-1,3-diene in wine.
A stable isotope dilution assay has been developed for quantification of (E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene (4) in wine using a [(2)H(6)]-analogue. Using this method, 4 was found in 96 out of 97 white wines, but in none of 12 red wines analyzed. 4 was found to be most prevalent in Semillon wines, followed by Chardonnay, with Riesling showing the least amount of 4 among these three varieties. 4, like 1,1,6-trimethyldihydronaphthalene (TDN, 3), appears to be formed during the aging process. 4 was found to be unstable in model wine, and in both white and red wine, with the order of stability being model > white > red. In a PVPP-stripped red wine, the rate of degradation of 4 was substantially lessened, with the final concentrations very close to those observed in model wine. When treated with either grape or wine tannin extracts in model wine, the concentration of 4 was found to decrease to levels very close to those observed with an untreated red wine. When white wine was heated at 45 degrees C, 4 was formed, indicating the presence of precursor forms. The amounts formed were much higher than those found in a commercial white wine. 4 was also observed in red wine heated to 45 degrees C, but the concentration produced was much less than that with white wine.
Alkenes↗
Personality and compatibility: a prospective analysis of marital stability and marital satisfaction.
The antecedents of marital stability (divorce or remaining married) and marital satisfaction (within the group that remains married) were investigated with a panel of 300 couples who were followed from their engagements in the 1930s until 1980. Twenty-two of the couples broke their engagements; of the 278 couples who married, 50 got divorced at some time between 1935 and 1980. Personality characteristics (measured by acquaintance ratings made in the 1930s) were important predictors of both marital stability and marital satisfaction. The three aspects of personality most strongly related to marital outcome were the neuroticism of the husband, the neuroticism of the wife, and the impulse control of the husband. In combination, the 17 major antecedent variables were moderately predictive of a criterion variable composed of both marital stability and marital satisfaction (R = .49). The three major aspects of personality accounted for more than half of the predictable variance. The remaining variance was accounted for by attitudinal, social-environment, and sexual history variables.
Divorce↗
Analysis of mRNA stabilities during pollen development and in BY2 cells
RNA stability is important in post-transcriptional gene expression. However, its role during plant male gametophyte development has not been studied in detail, probably because of technical difficulties and because earlier work suggested that, in general, mRNAs expressed during late pollen development would be stable. We determined mRNA stabilities of 12 pollen-expressed genes. We measured pollen mRNA decay during in planta development and in vitro maturation after transcriptional inhibition with ActinomycinD (ActD). Because five of these genes were also expressed in somatic cells we also measured RNA decay in BY2 cells after transcriptional inhibition with ActD. Ten of the mRNAs were highly stable during pollen development (t1/2 > 30 h), including all seven pollen-specific mRNAs. However, two mRNAs showed a logarithmic decrease as pollen development proceeded, both in planta and in vitro. In fact, for one of these two, GUT8-2b, the mRNA level decreased significantly within 3 h. Our results show that many pollen-expressed mRNAs are indeed long-lived but that at least some mRNAs undergo decay during pollen maturation. Thus, there is no overall cytoplasmic condition in pollen that stabilizes mRNAs and specific mRNA turnover mechanisms must exist. We detected reciprocal mRNA stabilities with two genes that are expressed in both BY2 cells and pollen. GUT15 mRNA, known to be unstable (t1/2 < 3 h) in BY2 cells, was highly stable in pollen. In contrast, GRP2 mRNA decayed in pollen but was highly stable in BY2 cells. Therefore, we conclude that mRNA stability can be tissue-specific.
Journal Article↗
The thermodynamic analysis of protein stabilization by sucrose and glycerol against pressure-induced unfolding.
We have studied the reaction native left arrow over right arrow denatured for the 33-kDa protein isolated from photosystem II. Sucrose and glycerol have profound effects on pressure-induced unfolding. The additives shift the equilibrium to the left; they also cause a significant decrease in the standard volume change (DeltaV). The change in DeltaV was related to the sucrose and glycerol concentrations. The decrease in DeltaV varied with the additive: sucrose caused the largest effect, glycerol the smallest. The theoretical shift of the half-unfolding pressure (P1/2) calculated from the net increase in free energy by addition of sucrose and glycerol was lower than that obtained from experimental mea- surements. This indicates that the free energy change caused by preferential hydration of the protein is not the unique factor involved in the protein stabilization. The reduction in DeltaV showed a large contribution to the theoretical P1/2 shift, suggesting that the DeltaV change, caused by the sucrose or glycerol was associated with the protein stabilization. The origin of the DeltaV change is discussed. The rate of pressure-induced unfolding in the presence of sucrose or glycerol was slower than the refolding rate although both were significantly slower than that observed without any stabilizers.
Glycerol↗
Group 13 grass allergens: structural variability between different grass species and analysis of proteolytic stability.
BACKGROUND: Determination of the allergen composition of an extract is essential for the improvement of hyposensitization therapy. Surprisingly, although grass pollen extracts have been studied intensively for 20 years, a further major allergen, Phl p 13, was detected recently in timothy grass pollen. OBJECTIVES: We sought to determine the occurrence and importance of group 13 allergens in various grass species and to investigate their proteolytic stability. METHODS: The group 13 allergens were determined by means of 2-dimensional PAGE blotting with patient sera and group 13-specific mAbs. The allergens were isolated chromatographically from several pollen extracts and analyzed by means of microsequencing. Cross-reactivity among various grass species was studied by using Western blots and immunoblot inhibition tests. The stability of the allergens was tested under defined extraction conditions. RESULTS: Group 13 allergens are detectable in all common grasses and show IgE cross-reactivity among them. The allergenic components were identified in the neutral pH range with molecular masses of 50 to 60 kd, and in the case of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5. Protein sequencing clearly confirms structural identities between different grass species, although individual variations are found. If low-molecular-mass components were depleted by means of gel filtration, a rapid degradation of group 13 allergens was observed. This is in contrast to other pollen allergens described thus far. CONCLUSION: Group 13 allergens are widespread and are major allergens in the grasses. Predicted from their primary structures, these allergens are polygalacturonases. This class of enzymes is already known from microorganisms, and these enzymes are recognized as potential inducers of asthma. Our studies indicate that the group 13 allergens show a considerable microheterogeneity and degradation, especially after depletion of low-molecular-mass components. One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.
Allergens↗
"Coarse" stability and bifurcation analysis using time-steppers: a reaction-diffusion example.
Evolutionary, pattern forming partial differential equations (PDEs) are often derived as limiting descriptions of microscopic, kinetic theory-based models of molecular processes (e.g., reaction and diffusion). The PDE dynamic behavior can be probed through direct simulation (time integration) or, more systematically, through stability/bifurcation calculations; time-stepper-based approaches, like the Recursive Projection Method [Shroff, G. M. & Keller, H. B. (1993) SIAM J. Numer. Anal. 30, 1099-1120] provide an attractive framework for the latter. We demonstrate an adaptation of this approach that allows for a direct, effective ("coarse") bifurcation analysis of microscopic, kinetic-based models; this is illustrated through a comparative study of the FitzHugh-Nagumo PDE and of a corresponding Lattice-Boltzmann model.
Computer Simulation↗
Analysis of thermal stabilizing interactions in mesophilic and thermophilic adenylate kinases from the genus Methanococcus.
Adenylate kinases (ADKs) from four closely related methanogenic members of the Archaea (the mesophile Methanococcus voltae (MVO), the thermopile Methanococcus thermolithotrophicus (MTH), and the extreme thermopiles Methanococcus igneus (MIG) and Methanococcus jannaschii (MJA)) were characterized for their resistance to thermal denaturation. Despite possessing between 68 and 81% sequence identity, the methanococcal ADKs significantly differed in their stability against thermal denaturation, with melting points ranging from 69 to 103 degrees C. The high sequence identity between these organisms allowed regions of the MVO and MJA ADKs to be exchanged, producing chimeric ADKs with significantly altered thermal stability. Up to a 20 degrees C increase or decrease in stability was achieved for chimeric ADKs, whereas 88% of the original protein sequence was maintained. Based on our previous structural modeling studies, we conclude that cooperative interactions within the hydrophobic protein core play an integral role in determining the differences in structural stability observed between the methanococcal ADKs. From comparisons of the effects of temperature on protein unfolding and optimal enzymatic activity, we also conclude that thermostability and enzymatic temperature optima are influenced differently by molecular modifications and thus that the protein flexibility required for activity and stability, respectively, is not unconditionally linked within the methanococcal ADKs.
Adenylate Kinase↗
Analysis of postural stability in collegiate soccer players before and after an acute bout of heading multiple soccer balls.
The aim of our study was to determine if any immediate changes in balance were discernable in college soccer players after a specially designed heading session. Eight male and two female skilled collegiate soccer players had a baseline balance pre-test using the Balance Master, followed by heading 20 balls kicked consecutively by a teammate from the touchline to a point near the goal, which was followed by a post-test using the same testing technique. Paired t-tests were used to compare balance ability between pre- and post-test conditions. There was no difference in balance pre- to post-test (85.3% and 86.2% respectively). No significant difference was noted from pre- to post-test in the mean equilibrium scores for conditions three through six on the Sensory Organization Test, with the exception of condition four, which revealed a significant increase from pre- to post-test. We concluded that an acute session of heading soccer balls may not result in balance changes in collegiate soccer players.
Adult↗
In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.
We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.
Animals↗