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PubMed1999

Bound volatile general anesthetics alter both local protein dynamics and global protein stability.

BACKGROUND: Recent studies have demonstrated that volatile general anesthetic agents such as halothane and isoflurane may bind to discrete sites on protein targets. In the case of bovine serum albumin, the sites of halothane and chloroform binding have been identified as being located in the IB and IIA subdomains. This structural information provides a foundation for more detailed studies into the potential mechanisms of anesthetic action. METHODS: The effect of halothane and isoflurane and the nonimmobilizer 1,2-dichlorohexafluorocyclobutane on the mobility of the indole ring in the tryptophan residues of albumin was investigated using measurements of fluorescence anisotropy. Myoglobin served as a negative control. In addition, the effect of bound anesthetic agents on global protein stability was determined by thermal denaturation experiments using near-ultraviolet circular dichroism spectroscopy. RESULTS: The fluorescence anisotropy measurements showed that halothane and isoflurane decreased the mobility of the indole rings in a concentration-dependent manner. The calculated dissociation constants were 1.6+/-0.4 and 1.3+/-0.3 mM for isoflurane and halothane, respectively. In contrast, both agents failed to increase the fluorescence anisotropy of the tryptophan residues in myoglobin, compatible with lack of binding. The nonimmobilizer 1,2-dichlorohexafluorocyclobutane caused no change in the fluorescence anisotropy of albumin. Binding of the anesthetic agents stabilized the native folded form of albumin to thermal denaturation. Analysis of the thermal denaturation data yielded dissociation constant values of 0.98+/-0.10 mM for isoflurane and 1.0+/-0.1 mM for halothane. CONCLUSIONS: Attenuation of local side-chain dynamics and stabilization of folded protein conformations may represent fundamental modes of action of volatile general anesthetic agents. Because protein activity is crucially dependent on inherent flexibility, anesthetic-induced stabilization of certain protein conformations may explain how these important clinical agents change protein function.

Algorithms↗
PubMedDate not supplied

Biomechanical comparison of a circular external skeletal fixator construct to pin and tension band wire fixation for the stabilization of olecranon osteotomies in dogs: a cadaveric study.

OBJECTIVE: To compare anatomic reduction and the biomechanical properties of a circular external skeletal fixator (CESF) construct to pin and tension band wire (PTBW) fixation for the stabilization of olecranon osteotomies in dogs. STUDY DESIGN: Cadaveric study. ANIMALS: Forelimbs from 12 skeletally mature mixed-breed dogs, weighing 23 to 28 kg. METHODS: An olecranon osteotomy was stabilized with either a CESF construct or PTBW fixation. A single distractive load to failure was applied to each specimen through the triceps tendon. Osteotomy reduction and biomechanical properties were compared between fixation groups. RESULTS: Reduction was not significantly different (gap: P =.171; malalignment: P =.558) between fixation groups. Osteotomies stabilized with the CESF had greater stiffness (P <.0001) and maximum load sustained (P <.0001) compared to PTBW fixation. There was no significant difference for yield load (P =.318) or for load at 1 mm of axial displacement (P =.997) between fixation groups. Failure of fixation occurred by bending of the intramedullary Steinmann pin and the fixation wires in the CESF specimens and by untwisting of the tension band wire knot with pullout and bending of the Kirschner wires in the PTBW specimens. CONCLUSIONS: Specimens stabilized with the CESF construct had similar reduction and yield load, greater stiffness and maximum load sustained, and less elastic deformation than specimens stabilized with PTBW fixation. CLINICAL RELEVANCE: The CESF construct may provide a biomechanically favorable alternative to PTBW fixation for stabilization of olecranon osteotomies in dogs, and its application warrants clinical investigation.

Animals↗
PubMed2005

Preparation and characterization of dehydration-rehydration vesicles loaded with aminoglycoside and macrolide antibiotics.

Enhanced activity of liposomes-encapsulated antibiotics against clinical isolates of Pseudomonas aeruginosa has been documented with liposomes of low encapsulation efficiency. We sought to construct liposomes with high yield entrapment of aminoglycoside and macrolide antibiotics as well as favorable stability in storage and physiological conditions. Liposome-entrapped aminoglycosides (amikacin, gentamicin, tobramycin) and a macrolide (erythromycin) were prepared by a modified dehydration-rehydration vesicles (DRVs) method, and their particle size and entrapment efficiency were determined. We studied in vitro stability of these vesicles over a 48 h period at 4 and 37 degrees C in phosphate-buffered saline (PBS) and in plasma at 37 degrees C. The mean particle size of DRVs loaded with antibiotics varied from 163.37+/-38.44 to 259.83+/-11.80 nm with no significant difference in regard with the type of the antibiotics encapsulated. Encapsulation efficiency of DRVs loaded with amikacin, gentamicin, tobramycin, and erythromycin were 29.27+/-1.17, 33+/-0.76, 22.33+/-1.48 and 32.06+/-0.82% of initial amount of the drug, respectively. These vesicles were stable regardless of the experimental temperature. Indeed, the liposomes retained more than 75% of the initially encapsulated drugs for the study period of 48 h. DRVs incubated in plasma however, released more antibiotics than those incubated in PBS. In conclusion, using this modified DRV method, we obtained small sized vesicles with high yield entrapment for aminoglycoside and macrolide antibiotics. The technique may be utilized to overcome the low encapsulation efficiency associated with aminoglycoside and macrolide antibiotics.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2006

p53 and p21 regulate error-prone DNA repair to yield a lower mutation load.

Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p21 to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p21. Loss of this regulation by inactivation of p53 or p21 causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability.

Animals↗
PubMed2006

Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins.

The branched sidechain residues 24 and 33 in the hydrophobic core of rubredoxin differ between the Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) sequences. Their X-ray structures indicate that these two sidechains are in van der Waals contact with each other, while neither appears to significantly interact with the other nonconserved residues. The simultaneous interchange of residues 24 and 33 between the Cp and Pf rubredoxin sequences yield a complementary pair of hybrid proteins for which the sum of their thermodynamic stabilities equals that of the parental rubredoxins. The 1.2 kcal/mol change arising from this two residues interchange accounts for 21% of the differential thermodynamic stability between the mesophile and hyperthermophile proteins. The additional interchange of the sole nonconserved aromatic residue in the hydrophobic core yields a 0.78 kcal/mol deviation from thermodynamic additivity.

Amino Acid Sequence↗
PubMed1999

Evidence for a proposed intermediate redox state in the CO/CO(2) active site of acetyl-CoA synthase (Carbon monoxide dehydrogenase) from Clostridium thermoaceticum.

When samples of the enzyme in the C(red1) state were reduced with Ti(3+) citrate, the C-cluster stabilized in an EPR-silent state. Subsequent treatment with CO or dithionite yielded C(red2). The EPR-silent state formed within 1 min of adding Ti(3+) citrate, while C(red2) formed after 60 min. Ti(3+) citrate appeared to slow the rate by which C(red2) formed from C(red1) and stabilize the C-cluster in the previously proposed C(int) state. This is the first strong evidence for C(int), and it supports the catalytic mechanism that required its existence. This mechanism is analogous to those used by flavins and hydrogenases to convert between n = 2 and n = 1 processes. Ti(3+) citrate had a different effect on enzyme in a CO(2) atmosphere; it shifted reduction potentials of metal centers (relative to those obtained using CO) and did not stabilize C(int). Different redox behavior was also observed when methyl viologen and benzyl viologen were used as reductants. This variability was exploited to prepare enzyme samples in which EPR from C(red2) was present without interfering signals from B(red). The saturation properties of B(red) depended upon the redox state of the enzyme. Three saturation "modes", called Sat1-Sat3, were observed. Sat1 was characterized by a sharp g = 1.94 resonance and low-intensity g = 2. 04 and 1.90 resonances, and was observed in samples poised at slightly negative potentials. Sat2 was characterized by weak intensity from all three resonances, and was strictly associated with intermediate redox states and the presence of CO(2). Sat3 was characterized by strong broad resonances with normalized intensities essentially unchanged relative to nonsaturating conditions, and was observed at the most negative potentials. Each mode probably reflects different spatial relationships among magnetic components in the enzyme.

Aldehyde Oxidoreductases↗
PubMed1994

On the relationship between the metabolic and thermodynamic stabilities of T4 lysozymes. Measurements in eukaryotic cells.

We have measured the metabolic stabilities of wild-type and 17 temperature-sensitive mutants of T4 lysozyme in HeLa cells, in Xenopus egg extract, and in reticulocyte lysate. [35S]Methionine-labeled T4 lysozymes were expressed in Escherichia coli, purified, injected into HeLa cells, and their degradation rates were determined. Wild-type T4 lysozyme has a half-life of 4 h; the half-lives of 16 lysozyme variants ranged from 2 to 10 h. Surprisingly, the most temperature-sensitive enzyme in the set, R96H, was significantly more stable (half-life = 10 h). Different T4 lysozyme variants yield conflicting answers to the proposed relationship between thermal and metabolic stabilities. For mutations at Thr157 there is no correlation between melting temperature and half-life. By contrast, T4 lysozymes mutated at various positions show a definite correlation between the two parameters. Treatment of injected HeLa cells with the lysosomotropic agents chloroquine or ammonium chloride did not alter the stability of T4 lysozyme. However, the enzyme's half-life increased 10-fold in HeLa cells depleted of ATP. Although T4 lysozyme is degraded rapidly within HeLa cells, the molecule is stable in reticulocyte lysate and Xenopus egg extract. Presumably, there is a specific proteolytic event(s) in HeLa cells which is not manifest in the in vitro extracts.

Animals↗
PubMed2005

Water-solubilized, cap-stabilized, helical polyalanines: calibration standards for NMR and CD analyses.

NMR and CD studies are reported for two length series of solubilized, spaced, highly helical polyalanines that are N-capped by the optimal helix stabilizer (beta)Asp-Hel and C-capped by beta-aminoalanine beta and that are studied in water at 2 degrees C, pH 1-8. NMR analysis yields a structural characterization of the peptide Ac(beta)AspHelAla(8)betaNH(2) and selected members of one (beta)AspHelAla(n)beta series. At pH > 4.5 the (beta)AspHel cap provides a preorganized triad of carboxylate anion and two amide residues that is complementary to the helical polyalanine N-terminus. The C-terminal beta-aminoalanine assumes a helix-stabilizing conformation consistent with literature precedents. H(N)CO NMR experiments applied to capped, uniformly (13)C- and (15)N-labeled Ala(8) and Ala(12) peptides define Ala(n) hydrogen bonding signatures as alpha-helical without detectable 3(10) character. Relative NH-->ND exchange rates yield site protection factors PF(i) that define uniquely high fractional helicities FH for the peptide Ala(n) regions. These Ala(n) calibration series, studied in water and lacking helix-stabilizing tertiary structure, yield the first (13)C NMR chemical shifts, (3)J(HNH)(alpha) coupling constants, and CD ellipticities [theta(Molar)](lambda,n) characteristic of a fully helical alanine within an Ala(n) context. CD data are used to assign parameters X and [theta](lambda,infinity), required for rigorous calculation of FH values from CD ellipticities.

Calibration↗
PubMed2002

Catalysis and stability of triosephosphate isomerase from Trypanosoma brucei with different residues at position 14 of the dimer interface. Characterization of a catalytically competent monomeric enzyme.

In homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM), cysteine 14 of each the two subunits forms part of the dimer interface. This residue is central for the catalysis and stability of TbTIM. Cys14 was changed to the other 19 amino acids to determine the characteristics that the residue must have to yield catalytically competent stable enzymes. C14A, C14S, C14P, C14T, and C14V TbTIMs were essentially wild type in activity and stability. Mutants with Asn, Arg, and Gly had low activities and stabilities. The other mutants had less than 1% of the activity of TbTIM. One of the latter enzymes (C14F) was purified to homogeneity. Size exclusion chromatography and equilibrium sedimentation studies showed that C14F TbTIM is a monomer, with a k(cat) approximately 1000 times lower and a K(m) approximately 6 times higher than those of TbTIM. In C14F TbTIM, the ratio of the elimination (methylglyoxal and phosphate formation) to isomerization reactions was higher than in TbTIM. Its secondary structure was very similar to that of TbTIM; however, the quantum yield of its aromatic residues was lower. The analysis of the data with the 19 mutants showed that to yield enzymes similar to the wild type, the residue must have low polarity and a van der Waals volume between 65 and 110 A(3). The results with C14F TbTIM illustrate that the secondary structure of TbTIM can be formed in the absence of intersubunit contacts, and that it has sufficient tertiary structure to support catalysis.

Animals↗
PubMed1993

Estimation of mitotic stability in conidial fungi: a theoretical framework.

Mitotic stability refers to the probability that genetic elements are transmitted to both daughters during mitosis. This is of practical importance in molecular genetics because autonomous cloning vectors should be transmitted at high frequency during mitosis. In filamentous coencytic fungi it is difficult to quantify mitotic stability because a fluctuation test is not feasible. We show how to get around this problem by formulating a general model of the transmission of nuclear genetic elements through the course of conidiogenesis. We derive formulas by two different methods for the expected proportion of conidiospores that retain the element as a function of its mitotic stability and the number of generations of spore production. An important by-product yields the exact probability distributions for the number of conidiospores retaining elements at each stage of conidiophore development. We outline, and illustrate through specific numerical examples, how to use these formulas to estimate mitotic stability. Although we use Aspergillus nidulans as our biological paradigm, the same general framework can be extended to other fungal species, and possibly to less closely related systems as well.

Aspergillus nidulans↗
PubMed1998

Volumetric properties of nucleic acids.

Volumetric studies can yield useful new information on a myriad of intra- and intermolecular interactions that stabilize nucleic acid structures. In particular, appropriately designed volumetric measurements can characterize the conformation-dependent hydration properties of nucleic acids as a function of solution conditions, including temperature, pressure, ionic strength, pH, and cosolvent concentration. We have started to accumulate a substantial database on volumetric properties of DNA and RNA, as well as on related low molecular weight model compounds. This database already has provided unique insights into the molecular origins of various nucleic acid recognition processes, including helix-to-coil and helix-to-helix conformational transitions, as well as drug-DNA interactions. In this article, we review recent progress in volumetric investigations of nucleic acids, emphasizing how these data can be used to gain insight into intra-and intermolecular interactions, including hydration properties. Throughout this review, we underscore the importance of volume and compressibility data for characterizing the hydration properties of nucleic acids and their constituents. We also describe how such volumetric data can be interpreted at the molecular level to yield a better understanding of the role that hydration can play in modulating the stability and recognition of nucleic acids.

Animals↗
PubMed1998

Prediction of the deficit syndrome from initial deficit symptoms in the early course of schizophrenia.

The Proxy for the Deficit Syndrome (PDS) was used with longitudinal symptom assessment data to identify recent-onset schizophrenia patients with the deficit syndrome. We evaluated the stability of deficit symptoms using repeated assessments. Symptom ratings were examined at an initial point of outpatient stabilization on antipsychotic medication as well as prospectively over the subsequent 12 months of outpatient treatment and assessment in 83 recent-onset schizophrenia patients. The vast majority of patients who were classified as non-deficit at the cross-sectional baseline assessment continued to remain non-deficit throughout the first year of treatment. However, patients classified as deficit at baseline did not consistently remain classified as showing deficit syndrome during the follow-through period. Thus, the presence of deficit symptoms detected in a single cross-sectional rating may be an inaccurate way to rate the deficit syndrome, yielding excessive false positives. Our use of longitudinal data allowed the stability criterion of the deficit syndrome to be evaluated using the PDS.

Adult↗
PubMed1991

Bacteriophage SPO1 middle transcripts.

Phage SPO1 middle transcripts are known to fall into two classes, m and m1l. Class m1l transcripts continue to be made late in the viral infection, while the synthesis of class m transcripts ceases soon after the onset of replication and late transcription. The experiments that are reported here deal with the regulatory nature of this diversity. The accumulation of transcripts associated with eight middle promoters was analyzed by S1 nuclease mapping. DNA sequence surrounding these middle promoters was determined or redetermined, and the stability of RNA associated with most of these promoters was also analyzed. Class m1l transcription was shown to be associated with SPO1 middle promoters that remain active at late stages of viral development, when middle promoters of class m are repressed. The consensus sequences of class m and m1l middle promoters were found to be indistinguishable and the search for sequences consensual with late promoters yielded only divergent candidates. No other consensus sequence that is specific and exclusive to either class of middle promoters was detected within a hundred base pairs upstream or downstream of these promoters. Considerable variations in the stabilities of SPO1 middle transcripts were found. Two promoters that are only 71 base pairs apart yielded transcripts that had substantially different stabilities. The 5'-flanking segment of the transcript associated with the upstream promoter apparently conferred a high degree of stability on this RNA.

Bacillus subtilis↗
PubMed1996

Stability of cereal allergens.

BACKGROUND: Ingested cereals and inhaled cereal dusts can cause IgE-mediated allergy. Testing for these allergies yields variable, often undependable results, which could be due to the use of unsuitable or degraded testing materials. OBJECTIVE: We studied the effect of storage media, time and temperature on the stability of protein allergens extracted from wheat, rye, barley and oats flour. We also examined the effect of heat processing similar to that used when preparing foods on the stability of allergenic proteins present in wheat flour. METHODS: After storage experiments proteins in allergen extracts were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then visualized using immunoblotting or staining with Coomassie brilliant blue. After heat processing wheat flour proteins were extracted and their IgE-binding capacity was estimated. RESULTS: Extracted proteins from wheat, rye and barley flour appeared stable for at least 21 months when stored at 4 degrees C in an aqueous solution containing 50% glycerol and 0.45% sodium chloride. In a medium with 0.4% phenol and 0.9% sodium chloride degradation of many proteins became apparent already after 1 month of storage. Oats extracts lost their IgE-binding capacity more rapidly and the maximal storage time appeared to be 10 months at 4 degrees C in a solution containing 50% glycerol and 0.45% sodium chloride. The IgE-binding capacity of the allergenic proteins decreased as heat-processing temperature and heating time increased. No processing conditions entirely abolished IgE-binding. CONCLUSION: Cereal allergen preparations can be stored either in lyophilized form or at 4 degrees C in 50% glycerol. Unheated materials should be used also when testing for food allergy, as heating reduces allergenicity and thus sensitivity at testing.

Adolescent↗
PubMed2002

Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.

Alcohols↗
PubMed1992

Anterior decompression, structural bone grafting, and Caspar plate stabilization for unstable cervical spine fractures and/or dislocations.

Fourteen patients who sustained acute cervical spine fractures and/or dislocations with associated posterior ligamentous disruption had anterior decompressions, structural bone grafting, and anterior Caspar plate stabilization. With an average 30-month follow-up, no patient has had loss of fixation. Despite criticism raised from biomechanical testing, the Caspar anterior plate system (Aesculape, Tuttlingen, Germany) may be added to structural bone grafting of unstable cervical fractures and/or dislocations, yielding an in vivo solid construct, which obviates the need for simultaneous posterior stabilization.

Adult↗
PubMed1998

A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing.

A library system was developed for the discovery of bioactive peptides. Library synthesis and peptide sequencing was performed on a solid support while the screening for bioactivity was done with peptides in solution. The peptides were synthesized by split and mix, one-bead-one-peptide library synthesis, using a Tentagel S-NH2 solid support with a loading of approximately 100 pmol/bead. The major part of the peptide was connected to the support by a single acid-labile linker and a minor part of the peptide was acid-stabile attached to the polymer. The percentage of acid-stabile attached peptides could easily be controlled during modification of the amino functionalities of the resin at the start of the process. The cleavage rate of the acid-labile attached peptide from the resin depends on the composition of the cleavage mixture. When cleavage conditions were carefully controlled, a three-step partial cleavage protocol allowed for convergent bioactivity screening on peptide libraries using only one type of acid-labile linker. The partial cleavage and convergent screening procedure was repeated three times, after which the bead containing the bioactive peptide was sequenced. As such a bead still contained acid-stabile attached peptide, the Edman sequencing was straightforward and repetitive yields were excellent because the immobilized peptide was not washed out.

CD4-Positive T-Lymphocytes↗
PubMed2000

Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila.

Drosophila melanogaster is an excellent system for examining the structure/function relationships of myosin. It yields insights into the roles of myosin in assembly and stability of myofibrils, in defining the mechanical properties of muscle fibers, and in dictating locomotory abilities. Drosophila has a single gene encoding muscle myosin heavy chain (MHC), with alternative RNA splicing resulting in stage- and tissue-specific isoform production. Localization of the alternative domains of Drosophila MHC on a three-dimensional molecular model suggests how they may determine functional differences between isoforms. We are testing these predictions directly by using biophysical and biochemical techniques to characterize myosin isolated from transgenic organisms. Null and missense mutations help define specific amino acid residues important in actin binding and ATP hydrolysis and the function of MHC in thick filament and myofibril assembly. Insights into the interaction of thick and thin filaments result from studying mutations in MHC that suppress ultrastructural defects induced by a troponin I mutation. Analysis of transgenic organisms expressing engineered versions of MHC shows that the native isoform of myosin is not critical for myofibril assembly but is essential for muscle function and maintenance of muscle integrity. We show that the C-terminus of MHC plays a pivotal role in the maintenance of muscle integrity. Transgenic studies using headless myosin reveal that the head is important for some, but not all, aspects of myofibril assembly. The integrative approach described here provides a multi-level understanding of the function of the myosin molecular motor.

Animals↗