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PubMed2006

Genetic analysis of the structure and function of transfer messenger RNA pseudoknot 1.

tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.

Amino Acid Sequence↗
PubMed1993

Application of a new statistical approach to optimize the immunopurification of antihemophilia A factor.

Our aim was to optimize the immunopurification process of human factor VIII. This purification was performed using a mouse monoclonal anti-factor VIII light-chain antibody. Previous dissociation of the factor VIII-von Willebrand factor complex with CaCl2 led to a 50% increase of the factor VIII adsorption on the immunosorbent. The optimization of the elution step required the analysis of the effects of two parameters, pH and ionic strength, on four different responses: elution yield, concentration, specific activity and stability of factor VIII. For this purpose, a multifunctional method using Doehlert matrices for statistically designed experiments was applied. This methodology allowed us to obtain, with only seven experiments, a 60% increase of the elution yield and a two-fold increase of the specific activity of factor VIII.

Antibodies, Monoclonal↗
PubMed1991

Encapsulation of doxorubicin in thermosensitive small unilamellar vesicle liposomes.

The optimisation of the formulation of thermosensitive, doxorubicin-containing small unilamellar liposomes is described. The liposomes were first strictly defined in terms of size distribution and size stability and a quality level was defined. The suspension contained more than 95% vesicles with a maximal diameter of 50 nm and kept this level for a minimum of 24 hours. Several lipid mixtures were tested in defined thermal conditions usable for in vitro experiments: 43 degrees C in fetal calf serum-containing medium. The mixture yielding the best differential thermal stability (DTS) defined as the difference of release between 37 degrees C and 43 degrees C exposures was found to be a dipalmitoylphosphatidylcholine/distearoylphosphatidyl-choline/chol esterol mixture in 5:4:2 molar ratio yielding 72% DTS. These thermosensitive liposomes were evaluated between pH 6.00 and 8.00 since hyperthermia-induced lethality was reported to be enhanced by pH variations. Their release capacity was not altered by any pH variations. Incorporation of doxorubicin within these liposomes was then performed. The release kinetics at 37 degrees and 43 degrees C were determined. It is proposed to use this formulation in in vitro experiments on tumour cells, although a decrease of DTS was evident.

Doxorubicin↗
PubMed2006

In vitro and in vivo evaluation of carbamazepine-loaded enteric microparticles.

The objective of the study was to prepare and evaluate carbamazepine-loaded enteric microparticles produced by a novel coacervation method. An aqueous polymeric stabilizer solution was added to an organic carbamazepine/Eudragit L100-55 solution. Water, which is a non-solvent for the drug and the enteric polymer, caused phase separation and the formation of coacervate droplets. These droplets hardened into microparticles upon further addition of the aqueous phase. The microparticles were characterized with respect to particle size distribution, morphology, encapsulation efficiency, yield, physical state and physical stability of the drug, wettability, in vitro release and in vivo bioavailability. Microparticles with a smooth surface and dense structure were obtained with high encapsulation efficiency (>85%) and yield (>90%). The drug was in a non-crystalline state in the matrix and physically stable for 5 months at room temperature. Under sink conditions, the drug dissolution rate from the microparticles was significantly enhanced compared to the physical mixture and to the pure drug; the release profile of the microparticles was stable after 5 months. Under non-sink conditions, an unstable supersaturated solution of carbamazepine was obtained from microparticles with the subsequent formation of needle-shaped crystals. The high surface area and good wettability of the microparticles, the non-crystalline state of the drug in the matrix and the fast dissolution rate contributed to a significantly enhanced oral bioavailability from the microparticles when compared to the physical mixture.

Acrylic Resins↗
PubMed2006

Triple helix stabilization by covalently linked DNA-bisbenzimidazole conjugate synthesized by maleimide-thiol coupling chemistry.

Tethering of BBZPNH2, an analogue of the Hoechst 33258, with a 14 nucleotide long DNA sequence with the help of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional crosslinking reagent, using DMF/ water as solvent yields a conjugate which effectively stabilizes the triple helix. The above conjugate was hybridized with 26 bp long double stranded (ds) DNA having 14 bp long polypurine-polypyrimidine stretch to form a pyrimidine motif triple helix. The above conjugate increases the thermal stability of both the transitions, that is, triple helix to double helix by 12 degrees C and double helix to single strand transition by 16 degrees C for the triple helix formed with conjugated TFO over the triple helix made from non-conjugated TFO. Fluorescence and circular dichroism spectra recorded at different temperatures confirm the presence of minor groove binding bisbenzimidazole in the AT-rich minor groove of dsDNA even after the major groove bound TFO separates out.

Benzimidazoles↗
PubMed1991

Continuous potable alcohol production by immobilized Saccharomyces cerevisiae on mineral kissiris.

A biocatalyst prepared by the immobilization of Saccharomyces cerevisiae on the surface of the mineral kissiris was used in the present study for continuous potable-alcohol production. An ethanol productivity (calculated on the basis of liquid volume) of 10.5 g/L/h was obtained at a 0.7/h dilution rate, 121 g/L sucrose content, and 29.6% conversion employing molasse as feed material. Glucose, raisin extracts, and molasse were successively used as feed materials without stopping the operation of the reactor for 6 mo. The ethanol productivity and yield remained constant during the operational-stability study of the reactor, carried out for 44 d. Biomass productivity, yield, and free-cell concentration in glucose, raisin extracts, and molasse were examined. Finally, a system with two continuous reactors joined successively was also studied in the present investigation.

Biotechnology↗
PubMed1995

Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog.

Recombinant DNA techniques now allow the production of "mini-antibodies" called Fv fragments. These have been produced either as the independent variable domains of the heavy and light chains non-covalently associated in one-to-one stoichiometry or as single-chain gene products with the two domains linked by an intervening peptide sequence. Although Fv fragments can have excellent binding properties, they are often difficult to produce in good yield and lack the characteristic stability of whole antibodies. To improve the stability of the Fv molecule, we have introduced a cysteine residue into conserved framework regions of both the heavy and light variable domains from the anti-Tac antibody at positions compatible with the formation of an interdomain disulfide linkage (i.e. VH-44 and VL-99). The mutant subunits form a disulfide-bonded Fv molecule, which binds to the alpha-subunit of the IL2 receptor (IL2R alpha) with an affinity identical to that of humanized anti-Tac IgG. This disulfide-stabilized Fv (dsFv) proved to be substantially more resistant to denaturation by heat or urea treatment than the single-chain Fv (scFv). Furthermore, the yield of dsFv is -four-fold higher than that of the single-chain analog.

Amino Acid Sequence↗
PubMed2001

Implementation of a chemical equilibrium constraint in the multivariate curve resolution of voltammograms from systems with successive metal complexes.

A multivariate curve resolution (MCR) method, using a constrained alternating least squares (ALS) procedure with a new chemical equilibrium constraint, was applied to differential-pulse polarograms of successive metal complexes. This new restriction imposes the fulfillment of a chemical model defined by a set of stability constants that are optimised along the iterative ALS procedure. The reliability of the method was tested with simulated data and with polarograms measured for the systems Zn(II) + glutathione and Cd(II) + 1,10-phenanthroline. These systems respectively yield two and three successive and electroactive complexes, which are inert from the electrochemical point of view, that is, the complexes virtually do not dissociate during the measurement. Although the presence of electrode adsorption could induce overestimation of some concentrations and losses of linearity between concentrations and signals, the results showed that the proposed method can yield satisfactory estimations of the stability constants in this kind of system. The performance of the new method is compared with the performances obtained using MCR-ALS without the equilibrium constraint and using traditional curve fitting least-squares approaches.

Journal Article↗
PubMed2005

Synthesis of isomeric angularly annealed dinaphthoporphyrin systems: examination of the relative positioning and orientation of ring fusion as factors influencing the porphyrin chromophore.

Porphyrins built up from two naphtho[1,2-c]pyrrole subunits and two beta-substituted pyrroles can produce five isomeric dinaphthoporphyrin systems. To gain insights into the effects of ring fusion on extended porphyrin chromophores, all five of these systems were synthesized in isomerically pure form. In four of these syntheses, dihydronaphthopyrroles were used to introduce one or both of the naphthalene subunits, and dehydrogenation with DDQ in refluxing toluene later produced the fully conjugated systems. Naphthopyrroles were also prepared by reacting isocyanoacetate esters with 1-nitronaphthalene in the presence of a phosphazene base. These compounds proved to be less stable than their dihydronaphthopyrrolic counterparts but could still be utilized in these synthetic studies. Three isomeric adj-dinaphthoporphyrin systems were prepared using the MacDonald "2 + 2" condensation or by the cyclization of a,c-biladiene intermediates with copper(II) chloride or AgIO(3)-Zn(OAc)(2). A dinaphthoporphyrin with two naphthalene units pointing toward one another could only be obtained in low yields due to a combination of stability and steric factors, but the other two adj-difused systems were isolated in good overall yields. However, the final dehydrogenation step occurred in moderate yields (50-60%) and could only be performed when the porphyrins bore propionate ester side chains that produced sufficient solubility in organic solvents. The two related opp-dinaphthoporphyrins were synthesized by a "head-to-tail" self-condensation of a dipyrrylmethane aldehyde, or a "3 + 1" synthesis using a tripyrrane intermediate bearing two fused dihydronaphthalene moieties, in excellent yields. In both cases, a final dehydrogenation step was required, but the opp-dinaphthoporphyrins were consistently formed in virtually quantitative yields. The opp-dinaphthoporphyrin series gave UV-vis spectra with relatively strong Soret bands at 425 nm, and the visible region was dominated by an unusually strong Q-band III. The adj-dinaphthoporphyrins produced broader less intense Soret bands and four well-defined Q-bands, including a relatively strong absorption at 645 nm. However, the relative orientation of the naphthalene rings had no significant effects on these spectra. On the other hand, the dications produced in TFA-chloroform solutions showed more discrimination between the individual porphyrin systems, and the metallo derivatives also displayed significant variations in their electronic absorption spectra.

Journal Article↗
PubMedDate not supplied

Importance of the matrix and its chemical activation on the stability of immuno-adsorbents.

Various matrices were reacted with four different activation reagents, in order to prepare immuno-adsorbents for the selective removal of low-density lipoproteins from blood plasma of patients affected by hypercholesterolaemia. The resulting immuno-adsorbents were compared to that obtained earlier with cyanogen bromide activation, in terms of coupling yield, adsorption capacity and moreover stability towards the various chemical and biochemical conditions to which they are submitted during their handling. Tresyl chloride-activated Sepharose 6 Fast Flow turns out to afford optimal stability in the whole pH range tested.

Chromatography, Affinity↗
PubMed2004

Stabilization of TRAIL, an all-beta-sheet multimeric protein, using computational redesign.

Protein thermal stability is important for therapeutic proteins, both influencing the pharmacokinetic and pharmacodynamic properties and for stability during production and shelf-life of the final product. In this paper we show the redesign of a therapeutically interesting trimeric all-beta-sheet protein, the cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), yielding variants with improved thermal stability. A combination of tumor necrosis factor (TNF) ligand family alignment information and the computational design algorithm PERLA was used to propose several mutants with improved thermal stability. The design was focused on non-conserved residues only, thus reducing the use of computational resources. Several of the proposed mutants showed a significant increase in thermal stability as experimentally monitored by far-UV circular dichroism thermal denaturation. Stabilization of the biologically active trimer was achieved by monomer subunit or monomer-monomer interface modifications. A double mutant showed an increase in apparent T(m) of 8 degrees C in comparison with wild-type TRAIL and remained biologically active after incubation at 73 degrees C for 1 h. To our knowledge, this is the first study that improves the stability of a large multimeric beta-sheet protein structure by computational redesign. A similar approach can be used to alter the characteristics of other multimeric proteins, including other TNF ligand family members.

Algorithms↗
PubMed1989

An improved noninvasive method for measurement of cardiac output and evaluation of left-sided cardiac valve incompetence.

A time-saving method was developed to label red blood cells in vitro with 99mTc while avoiding centrifugation. After tin incubation, extracellular tin was oxidized by sodium hypochlorite, and EDTA was added for stabilizing the complex prior to 99mTc incubation. Labeling yields were 95%, and in vivo decay showed a high stability with a mean biologic half-life of eleven hours. The first-passage radionuclide technique for determination of cardiac output using the above-mentioned tracer was evaluated by using the left ventricle as area-of-interest with individual background correction after complete mixing of the tracer. This technique showed a high level of agreement with invasive methods. By combining this method for measurement of the forward stroke volume with the multigated equilibrium principle for determination of the total left ventricular stroke volume using similar background corrections, an exact evaluation of regurgitation fractions was obtained. In patients with aortic and mitral valve disease the noninvasive radionuclide technique gave similar but probably more accurate results as compared with contrast aortography and ventriculography. The radionuclide technique may be suitable for monitoring and selecting patients for surgical treatment.

Aortic Valve Insufficiency↗
PubMed2004

Use of trial clustering to study QTL x environment effects for grain yield and related traits in maize.

A population of 300 F(3:4) lines derived from the cross between maize inbred lines F2 and F252 was evaluated for testcross value in a large range of environmental conditions (11 different locations in 2 years: 1995 and 1996) in order to study (1) the magnitude of genotype x environment and (2) the stability of quantitative trait loci (QTL) effects. Several agronomic traits were measured: dry grain yield (DGY), kernel weight, average number of kernels per plant, silking date (SD) and grain moisture at harvest. A large genotype x environment interaction was found, particularly for DGY. A hierarchical classification of trials and an additive main effects and multiplicative interaction (AMMI) model were carried out. Both methods led to the conclusion that trials could be partitioned into three groups consistent with (1) the year of experiment and (2) the water availability (irrigated vs non-irrigated) for the trials sown in 1995. QTL detection was carried out for all the traits in the different groups of trials. Between 9 and 15 QTL were detected for each trait. QTL x group and QTL x trial effects were tested and proved significant for a large proportion of QTL. QTL detection was also performed on coordinates on the first two principal components (PC) of the AMMI model. PC QTL were generally detected in areas where QTL x group and QTL x trial interactions were significant. A region located on chromosome 8 near an SD QTL seemed to play a key role in DGY stability. Our results confirm the key role of water availability and flowering earliness on grain yield stability in maize.

Chromosome Mapping↗
PubMed2006

In situ temperature-time effect on MetA-S-SIMS.

Metal-assisted (MetA) static secondary ion mass spectrometry (S-SIMS) is one of several ion yield enhancing methods developed for S-SIMS in the last decades. MetA-S-SIMS uses a very thin coating of gold or silver on the sample. Earlier experiments revealed dependence of the ion yield enhancement on the applied metal, the nature of the studied sample, the time after metallization, and the heating temperature (ex situ, i.e., under atmospheric pressure). This paper reports on the effects of time and temperature when samples are heated to temperatures between 30 and 80 degrees C inside the S-SIMS vacuum chamber (in situ). Thick layers of poly(vinylbutyral-co-vinylalcohol-co-vinylacetate) (PVB) containing dihydroxybenzophenone (DHBPh) were coated with a nm-thin-layer of gold. The S-SIMS analysis was performed over a period of several hours while samples were kept at a constant elevated temperature. Compared to ex situ heating in an oven, heating in the analysis chamber provided more rapid signal enhancement, but the magnitude of the enhancement was less (by a factor of two). Furthermore, additional experiments on ex situ heated samples revealed that storage of samples with enhanced ion yields at -8 degrees C is not sufficient to "stabilize" the enhancement. A steep decrease of the ion yields was observed as a function of time after 2.5 h.

Journal Article↗
PubMed1994

Domain interactions and connecting peptides in lens crystallins.

beta B2- and gamma B-crystallin from bovine eye-lens are closely related proteins, topologically distinct mainly by virtue of the linker peptide connecting the two domains in each polypeptide chain. In homodimeric beta B2-crystallin, the extended conformation of the connecting peptide has been suggested to force the beta B2-molecule to favor intermolecular domain interactions compared with intramolecular contacts in monomeric gamma B-crystallin. From this one may postulate that the conserved interdomain contacts are essential for the overall stability of crystallins. This was clearly confirmed for gamma B-crystallin, since its isolated C-terminal domain is significantly less stable than in the context of native gamma B. Exchanging the linker peptide of gamma B- for that of beta B2-crystallin yields a monomeric protein with stability characteristics identical to gamma B-crystallin. We conclude that the domain-interface itself rather than the connecting peptide determines the mode of domain association in crystallins, as the linker in the gamma B beta-mutant is evidently twisted to a turn similar to the one in natural gamma B-crystallin.

Amino Acid Sequence↗
PubMed2004

Hydrolytic stability of organic monolayers supported on TiO2 and ZrO2.

The hydrolytic stability of C18 monolayers supported on TiO2 and ZrO2 was studied. Three types of monolayers were prepared from the following octadecyl modifiers: (1) octadecyldimethylchlorosilane (C18H37Si(CH3)2Cl); (2) octadecylsilane (C18H37SiH3); and (3) octadecylphosphonic acid (C18H37P(O)(OH)2). The hydrolysis of the surfaces prepared was studied under static conditions at 25 and 65 degrees C at pH 1-10. On the basis of the loss of grafted material, the stability of the monolayers fall in the following range: C18H37P(O)(OH)2 > or = C18H37SiH3 >> C18H37Si(CH3)2Cl. At 25 degrees C, monolayers from C18H37P(O)(OH)2 showed only approximately 2-5% loss in grafting density after one week at pH 1-10. The high stability of these monolayers was explained because of the strong interactions of the phosphonic acids with the substrates. Monolayers from C18H37Si(CH3)2Cl showed poor hydrolytic stability at any pH, which was explained because of the low stability of Ti-O-Si and Zr-O-Si bonds. Unlike monofunctional silanes, trifunctional silane (C18H37SiH3) yielded surfaces that showed good hydrolytic stability. This suggests that the stability of the monolayers from trifunctional silanes is primarily due to "horizontal" bonding (Si-O-Si or Si-OH...HO-Si) rather than due to bondingwith the matrix (M-O-Si). At 65 degrees C, all C18 surfaces become more susceptible to hydrolysis; however, the trend observed for 25 degrees C remained unchanged. Low-temperature nitrogen adsorption was used to study the adsorption properties of the monolayers as a function of their grafting density. The energy of adsorption interactions showed a significant increase as the grafting density of the monolayers decreased. The order of the alkyl groups in the monolayers, as assessed from CH2 stretching, decreased as the grafting density of the monolayers decreased.

Journal Article↗
PubMed1989

On the degree of stability of measured hypnotizability over a 25-year period.

Conducted a longitudinal study of hypnotizability, as measured by the Stanford Hypnotic Susceptibility Scale, Form A, that yielded a relatively high degree of stability in hypnotic responsiveness over repeated testings spanning a 25-year period. The 50 Ss were retested in 1985, after tests when they were students, between 1958-1962 and again in 1970. The statistically significant stability coefficients were .64 (10-year retest), .82 (15-year retest), and .71 (25-year retest). The means did not change significantly, and the median change in the scores of individuals was only 1 point on the 12-item scale. A set of score measures and their intercorrelations are insufficient to resolve the issue of why stability occurs. The stability of hypnotizability over time compares favorably with that of other measures of individual differences.

Adult↗