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PubMedDate not supplied

Stable, long-term bacterial production of soluble, dimeric, disulfide-bonded protein pharmaceuticals without antibiotic selection.

Numerous biopharmaceuticals and other recombinant biotechnology products are made in prokaryotic hosts. However, bacterial production of native, biologically active eukaryotic proteins is rarely possible for disulfide-bonded and/or multisubunit proteins. We previously described the production of soluble, native disulfide-bonded dimeric proteins in the Escherichia coli cytoplasm (Miele et al., 1990; Mantile et al., 1993). Native, biologically active proteins with up to six disulfide bonds have been produced with our expression system (Garces et al., 1997). However, plasmid instability during induction limited its usefulness. We now report the stable, high-level expression of soluble, disulfide-bonded human uteroglobin without antibiotic selection. We designed a new vector containing a multifunctional stabilization region that confers complete plasmid stability and increased protein yields without copy number increases. Recombinant expression remains fully inducible after long-term continuous culture in nonselective liquid medium (at least 260 generations). This system may significantly expand the applications of bacterial expression to recombinant production of soluble, bioactive proteins for biochemical studies and biopharmaceutical/industrial purposes. As a result of the very broad activity spectrum of the stabilization region we selected, its use could be extended to bacterial hosts other than enterobacteria.

Anti-Bacterial Agents↗
PubMed2002

Non-standard insulin design: structure-activity relationships at the periphery of the insulin receptor.

The design of insulin analogues has emphasized stabilization or destabilization of structural elements according to established principles of protein folding. To this end, solvent-exposed side-chains extrinsic to the receptor-binding surface provide convenient sites of modification. An example is provided by an unfavorable helical C-cap (Thr(A8)) whose substitution by favorable amino acids (His(A8) or Arg(A8)) has yielded analogues of improved stability. Remarkably, these analogues also exhibit enhanced activity, suggesting that activity may correlate with stability. Here, we test this hypothesis by substitution of diaminobutyric acid (Dab(A8)), like threonine an amino acid of low helical propensity. The crystal structure of Dab(A8)-insulin is similar to those of native insulin and the related analogue Lys(A8)-insulin. Although no more stable than native insulin, the non-standard analogue is twice as active. Stability and affinity can therefore be uncoupled. To investigate alternative mechanisms by which A8 substitutions enhance activity, multiple substitutions were introduced. Surprisingly, diverse aliphatic, aromatic and polar side-chains enhance receptor binding and biological activity. Because no relationship is observed between activity and helical propensity, we propose that local interactions between the A8 side-chain and an edge of the hormone-receptor interface modulate affinity. Dab(A8)-insulin illustrates the utility of non-standard amino acids in hypothesis-driven protein design.

Amino Acid Sequence↗
PubMed2004

Rational engineering of enzyme stability.

During the past 15 years there has been a continuous flow of reports describing proteins stabilized by the introduction of mutations. These reports span a period from pioneering rational design work on small enzymes such as T4 lysozyme and barnase to protein design, and directed evolution. Concomitantly, the purification and characterization of naturally occurring hyperstable proteins has added to our understanding of protein stability. Along the way, many strategies for rational protein stabilization have been proposed, some of which (e.g. entropic stabilization by introduction of prolines or disulfide bridges) have reasonable success rates. On the other hand, comparative studies and efforts in directed evolution have revealed that there are many mutational strategies that lead to high stability, some of which are not easy to define and rationalize. Recent developments in the field include increasing awareness of the importance of the protein surface for stability, as well as the notion that normally a very limited number of mutations can yield a large increase in stability. Another development concerns the notion that there is a fundamental difference between the "laboratory stability" of small pure proteins that unfold reversibly and completely at high temperatures and "industrial stability", which is usually governed by partial unfolding processes followed by some kind of irreversible inactivation process (e.g. aggregation). Provided that one has sufficient knowledge of the mechanism of thermal inactivation, successful and efficient rational stabilization of enzymes can be achieved.

Biotechnology↗
PubMed2001

Simple and reliable preparation of pentavalent 99Tcm-dimercaptosuccinic acid at alkaline pH without oxygen bubbling.

We investigated a simple and reliable method for the preparation of 99Tcm-dimercaptosuccinic acid (99Tcm(V)-DMSA) without the addition of oxygen. The effect of pH, amount of reducing agent, and oxygen addition in the synthesis of 99Tcm(V)-DMSA were evaluated. At pH 9, we obtained a radiochemical yield of 95% +/- 1.2% within 10 min and a high stability until 7 h, with 92% +/- 1.5% radiochemical purity. However, at a pH lower than 9, the radiochemical yield was below 90% within 10 min, and a longer reaction time was needed to obtain a radiochemical yield above 90%. The addition of oxygen did not have an additional effect on the radiochemical yield or its stability at pH 9, whereas it increased the radiochemical yield of 99Tcm-(V)-DMSA at pH 7. It was noted that the smaller the amount of reducing agent used, the higher was the radiochemical yield obtained at pH 7. However, at pH 9, the radiochemical yield was not dependent on the amount of reducing agent. In conclusion, the synthesis of 99Tcm(V)-DMSA was more dependent on the pH of the reaction mixture than on the amount of reducing agent or the addition of oxygen. The adjustment of pH 9 was the easiest and most effective method for the synthesis of 99Tcm(V)-DMSA using a commercial kit for 99Tcm(III)-DMSA.

Drug Compounding↗
PubMed1994

Effects of postirradiation temperature on the yields of radiation-induced single- and double-strand breakage in SV40 DNA.

The effects of postirradiation holding temperature on the yields of radiation-induced single- and double-strand breaks (SSBs and DSBs) in SV40 DNA have been measured by agarose gel electrophoresis. When the DNA is held at low temperatures (< or = 2 degrees C) before and during electrophoresis, the measured yields of radiation-induced SSBs and DSBs are twofold less than in samples exposed to room temperature. In contrast, if the DNA is incubated at 37 degrees C overnight, the yield of DSBs increases twofold over the room temperature assay, while the SSB yield increases only to a small extent (< or = 20%). From a comparison of the various yields, we suggest that low temperature stabilizes radiation-induced labile sites, and that the increased yield of DSBs at 37 degrees C is due either to the recruitment of spatially separate SSBs as DSBs by duplex melting, or to labile sites generating DSBs. The different routes to DSB formation are kinetically distinct. We conclude that room-temperature electrophoresis measures all SSBs including those from labile sites.

Cell Line↗
PubMed1992

Operational stability of enzymes. Acylase-catalyzed resolution of N-acetyl amino acids to enantiomerically pure L-amino acids.

The method of measuring enzyme deactivation by monitoring necessary addition of fresh enzyme to keep a constant degree of conversion in a CSTR at constant [E] x tau, the product of concentration of active enzyme [E] and residence time tau, was successfully applied to acylase I from porcine kidney and Aspergillus oryzae fungus. Fungal enzyme was found to be more stable than kidney enzyme. Activation by both Co2+ and Zn2+ ions also yielded increased operational enzyme stability: Co2+ and Zn2+ are better stabilizers than activators. Mg2+ and Ca2+ are found to be neither activators nor stabilizers. Fungal acylase partially deactivated by exposition to a metal-free medium in the CSTR was reactivated by addition of Zn2+, demonstrating that loss of Zn2+ from the enzyme molecule is mainly responsible for deactivation in a continuous reactor.

Amidohydrolases↗
PubMed1985

Enzymatic and nonenzymatic dehydration reactions of L-arogenate.

L-Arogenate, an immediate precursor of either L-tyrosine, L-phenylalanine, or both in many microorganisms and plants, may undergo two types of dehydration reactions that yield products of increased stability. Under acidic conditions, a facile aromatization attended by loss of the C-4 hydroxyl and the C-1 carboxyl moieties results in quantitative conversion to L-phenylalanine. When aromatization was largely prevented by maintaining pH in the range of 7.5-12, a second dehydration reaction occurred in which the alanyl side chain and the carboxyl group at C-1 formed a lactam ring to yield spiro-arogenate. The latter reaction occurs at 100 degrees C, roughly 50% conversion being obtained in 2 h. The product formed from L-arogenate was authentic spiro-arogenate, as demonstrated by high-performance liquid chromatography and thin-layer chromatography identification procedures. Further confirmation was obtained by 1H nuclear magnetic resonance, ultraviolet spectroscopy, and mass spectrometry. Thus far, the conversion of L-arogenate to spiro-arogenate is not known to be enzyme catalyzed. The other dehydratase reaction, however, is catalyzed in nature by an enzyme denoted arogenate dehydratase. An improved assay is described for this in which [3H]dansyl derivatives of L-arogenate (substrate) and L-phenylalanine (product) are separated by using bidimensional thin-layer chromatography. The radioactive reaction product is then quantitated. This assay was used to study partially purified arogenate dehydratase from Pseudomonas diminuta, an organism that depends upon the arogenate pathway for L-phenylalanine biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids, Dicarboxylic↗
PubMed2001

V490M, a common mutation in 3-phosphoglycerate dehydrogenase deficiency, causes enzyme deficiency by decreasing the yield of mature enzyme.

A deficiency of 3-phosphoglycerate dehydrogenase (PHGDH) is a disorder of serine biosynthesis identified in children with congenital microcephaly, seizures, and severe psychomotor retardation. We report here the identification of the 1468G-->A (V490M) mutation of this gene in two siblings of an Ashkenazi Jewish family, providing further evidence that the V490M mutation is a common, panethnic cause of this deficiency. Using a novel, DNA-based diagnostic test, the mutation was not detected in 400 non-Jewish controls; one heterozygote was found among 400 persons of Ashkenazi Jewish ethnicity. Extensive biochemical studies were undertaken to characterize the effect of this mutation on enzyme activity, turnover, and stability. The V490M PHGDH yielded less than 35% of the activity observed for the wild-type enzyme when overexpressed by transient transfection or when comparing the endogenous activity in fibroblast cells from the patients with controls. Immunoblotting studies showed a comparable reduction in the level of immunoreactive PHGDH in cells expressing the mutant enzyme. Pulse-chase experiments with metabolically labeled PHGDH indicated that this resulted from an increased rate of degradation of the mutant enzyme following its synthesis. Thermolability analyses of mutant and wild-type enzyme activity revealed no significant differences. While others have proposed that the V490M mutation decreases the V(max) of the enzyme, we conclude that this mutation impairs the folding and/or assembly of PHGDH but has minimal effects on the activity or stability of that portion of the V490M mutant that reaches a mature conformation.

Animals↗
PubMed2000

Chronic fatigue syndrome: an examination of the phases.

The present study examined the Fennell Phase Inventory, an instrument designed to measure the phases typically experienced by individuals with chronic fatigue syndrome (CFS). This inventory yields three factor scores of Crisis, Stabilization, and Integration. These factor scores have been employed in a cluster analysis, yielding four clusters that matched the four phases predicted by Fennell: Crisis, Stabilization, Resolution, and Integration. The present study represents a partial replication study of a prior investigation of the Fennell Phase Inventory by Jason et al. (in press), but that earlier study did not have an independent physician examination to diagnose patients with CFS. In the present study, 65 patients diagnosed with chronic fatigue syndrome by a physician were recruited and administered the Fennell Phase Inventory and other measures assessing CFS-related symptoms, disability, and coping. Each of the 65 patients was classified into one of four predefined clusters measuring a Crisis phase, a Stabilization phase, a Resolution phase, and an Integration phase. Relationships were explored between three of these cluster groupings and measures of symptoms, disability, and coping. Results confirmed Fennell's model, revealing significant differences between the three clusters in terms of levels of disability and modes of coping. Results suggest that the Fennell Phase Inventory accurately differentiates phases of adaptation to illness experienced by individuals with CFS.

Adaptation, Psychological↗
PubMed2005

Choosing your implant: cemented, tricompartmental, and posterior stabilized.

Cemented, posterior-stabilized, tricompartmental implants have yielded excellent relief of pain and are applicable to almost all clinical situations. The complications and revision rates after routine patellar resurfacing are less than those seen without such resurfacing. Cine fluoroscopic studies have shown that they restore a more normal kinematic pattern than do posterior cruciate ligament-retaining implants and have a low rate of radiographic and/or clinical loosening when followed up through 15 years after surgery. Latest posterior stabilized designs can allow a higher degree of flexion than seen in any other combination of implant designs. It is for all these reasons that the author feels that this method of performing a knee arthroplasty is the gold standard.

Arthroplasty, Replacement, Knee↗
PubMed2004

Electro-oxidation and amperometric detection of chlorinated phenols at boron-doped diamond electrodes: a comparison of microcrystalline and nanocrystalline thin films.

We report on the electro-oxidation and amperometric detection of phenol and chlorinated phenols, the latter coupled with flow injection analysis (FIA) and high performance liquid chromatography (HPLC), using boron-doped microcrystalline and nanocrystalline diamond thin-film electrodes. The low background current, good response without extensive pretreatment, and low susceptibility to fouling are properties that make diamond an attractive new electrode for monitoring this class of pollutants. Cyclic voltammetric studies were performed to evaluate the redox response of phenol, 2-chlorophenol, 3-chlorophenol,4-chlorophenol, and pentachlorophenol (PCP) in phosphate buffer, pH 3.5, as a function of the potential scan rate and cycle number. The diamond electrode performance for the amperometric detection of these contaminants in FIA-EC and HPLC-EC was evaluated in terms of the linear dynamic range, limit of quantitation, sensitivity, response precision, and response stability. Both diamond types yielded low mass limits of quantitation of 100-1000 pg for all the phenolic compounds in FIA-EC, except PCP which was 3 ng, and 100-600 pg for all the compounds in HPLC-EC. In all cases, the S/N was 3 or greater. Both electrode types also exhibited good sensitivity, excellent response reproducibility (av 2.7% for FIA-EC and av 4.2% for HPLC-EC), and superb response stability for all the analytes. The electrodes could be used from days to weeks in the measurement with only a periodic soak in distilled 2-propanol required to maintain optimum performance. Both types of diamond outperformed glassy carbon, which exhibited short-lived responsiveness as a consequence of fouling by reaction products and potential-dependent changes in the electrode's physiochemical properties. The use of the HPLC-EC assay for the determination of 2-chlorophenol in a contaminated soil sample is also demonstrated.

Boron↗
PubMed1997

Ion Adsorption and Agglomeration Mechanism in Si3N4/H2O(l) Dispersions Compatible with Thermodynamical Stability

Adsorption isotherms from aqueous liquid phase of H+ and/or OH- ions onto Si3N4 solid surface agglomerates have been derived through titration measurements (Beruto et al., J. Chem. Soc., Faraday Trans. 91(2), 323 (1995)). The isotherms exhibit S-shape and can be fitted with Freundlich as well as with Langmuir type isotherms. In order to explain the agglomeration process based on the decrease of total solid-liquid interfacial energy, a model (ISD) has been developed. The agreement between numerical data derived by this model and the experimental data is quite good. Isotherm equation and agglomeration law should obey also the dispersion thermodynamic stability criterion. This condition yields an extra equation to select the pair of functions, thus providing a criterion to choose between the several equations that fit adsorption and agglomeration data fairly well. The solution of the problem mentioned above, from the mathematical point of view, is based on the Dini implicit function theorem (Mezzasalma and Oliva, Appl. Math. Lett., submitted). The results indicate that Van der Waals adsorption isotherm coupled with the ISD model are compatible with the thermodynamic stability of the aqueous Si3N4 dispersions.

Journal Article↗
PubMedDate not supplied

The Perceived Stress Questionnaire (PSQ) reconsidered: validation and reference values from different clinical and healthy adult samples.

OBJECTIVE: The aim was to translate, revise, and standardize the Perceived Stress Questionnaire (PSQ) by Levenstein et al. (1993) in German. The instrument assesses subjectively experienced stress independent of a specific and objective occasion. METHODS: Exploratory factor analyses and a revision of the scale content were carried out on a sample of 650 subjects (Psychosomatic Medicine patients, women after delivery, women after miscarriage, and students). Confirmatory analyses and examination of structural stability across subgroups were carried out on a second sample of 1,808 subjects (psychosomatic, tinnitus, inflammatory bowel disease patients, pregnant women, healthy adults) using linear structural equation modeling and multisample analyses. External validation included immunological measures in women who had suffered a miscarriage. RESULTS: Four factors (worries, tension, joy, demands) emerged, with 5 items each, as compared with the 30 items of the original PSQ. The factor structure was confirmed on the second sample. Multisample analyses yielded a fair structural stability across groups. Reliability values were satisfactory. Findings suggest that three scales represent internal stress reactions, whereas the scale "demands" relates to perceived external stressors. Significant and meaningful differences between groups indicate differential validity. A higher degree of certain immunological imbalances after miscarriage (presumably linked to pregnancy loss) was found in those women who had a higher stress score. Sensitivity to change was demonstrated in two different treatment samples. CONCLUSION: We propose the revised PSQ as a valid and economic tool for stress research. The overall score permits comparison with results from earlier studies using the original instrument.

Abortion, Spontaneous↗
PubMed1989

Experimental method for the in vitro testing of the initial stability of cementless hip prostheses.

Micromotions at the interface between bone and prosthesis are believed to induce bone resorption and ultimately lead to loosening of the implant. Thus the initial stability achieved by a hip prosthesis is an important factor for the long-term function of the implant. Knowing the biological consequences of the mechanical conditions, it appears to be mandatory to measure the extent of these three-dimensional movements. An in vitro dynamic method for measurement of the micromotion of the femoral component of hip prostheses has been developed. Tests in cemented prostheses have confirmed that the use of cement reduces sinkage and rotation manyfold and have yielded reference values for stability. Comparison with two types of cementless prostheses has shown that certain cementless implants may achieve stability comparable to cemented ones in some load directions.

Bone Cements↗
PubMed2003

Synthesis of a Möbius aromatic hydrocarbon.

The defining feature of aromatic hydrocarbon compounds is a cyclic molecular structure stabilized by the delocalization of pi electrons that, according to the Hückel rule, need to total 4n + 2 (n = 1,2, em leader ); cyclic compounds with 4n pi electrons are antiaromatic and unstable. But in 1964, Heilbronner predicted on purely theoretical grounds that cyclic molecules with the topology of a Möbius band--a ring constructed by joining the ends of a rectangular strip after having given one end half a twist--should be aromatic if they contain 4n, rather than 4n + 2, pi electrons. The prediction stimulated attempts to synthesize Möbius aromatic hydrocarbons, but twisted cyclic molecules are destabilized by large ring strains, with the twist also suppressing overlap of the p orbitals involved in electron delocalization and stabilization. In larger cyclic molecules, ring strain is less pronounced but the structures are very flexible and flip back to the less-strained Hückel topology. Although transition-state species, an unstable intermediate and a non-conjugated cyclic molecule, all with a Möbius topology, have been documented, a stable aromatic Möbius system has not yet been realized. Here we report that combining a 'normal' aromatic structure (with p orbitals orthogonal to the ring plane) and a 'belt-like' aromatic structure (with p orbitals within the ring plane) yields a Möbius compound stabilized by its extended pi system.

Journal Article↗
PubMed1992

Parenchymal stability.

Both continuum and micromechanical models have been used to describe the mechanics of lung parenchyma. Different authors, using different models, have come to different conclusions about parenchymal stability. We show that the continuum model, augmented by bounds on the elastic moduli obtained from recent micromechanical modeling, yields the same conclusions about stability that have been obtained from purely micromechanical modeling: if the lung were homogeneous, it would be stable; local atelectasis would not occur at positive transpulmonary pressure. However, the same analysis yields the prediction that if the surface-to-volume ratio is not uniform throughout the lung, regions of higher surface density collapse if surface tension is large and insensitive to surface area. A micromechanical model that illustrates regional collapse is described.

Humans↗
PubMedDate not supplied

New coupling reagents for the preparation of disulfide cross-linked conjugates with increased stability.

To improve the in vivo stability of disulfide-linked immunotoxins (ITs), a series of sterically hindered cross-linking reagents were designed and synthesized. These ligands are characterized by a thioimidate group linked to an S-acetyl thiol or a substituted aryldithio group. To select the reagent of choice, several aryldithio thioimidates, substituted with a methyl or a phenyl group adjacent to the disulfide, were analyzed in thiol-disulfide exchange reactions. Also analyzed were the following: (i) the stability and solubility of the linkers in aqueous solution, (ii) the rate of protein derivatization, and (iii) the steric hindrance due to methyl or phenyl group substituents toward cleavage of the disulfide bond by glutathione. Ethyl S-acetyl 3-mercaptobutyrothioimidate (M-AMPT) was chosen as reagent to prepare two types of stable disulfide-containing AR-3-gelonin conjugates (IT2 and IT3). IT2 was prepared by a 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT)-derivatized antibody coupled to the M-AMPT-derivatized gelonin to afford a conjugate characterized by the presence of a methyl group adjacent to the sulfide bond. In the IT3 conjugate, an M-AMPT-derivatized toxin was coupled to the antibody thiolated with M-AMPT and then activated with Ellman's reagent (DNTB). The in vitro and in vivo stabilities of the three immunoconjugates were assayed, respectively, (i) by adding an excess of glutathione and monitoring protein release and (ii) by studying their pharmacokinetic behaviors. The specificity and cytotoxicity of all ITs were analyzed on target and unrelated cell lines, and no significant differences in activity were observed. IT3, consisting of a symmetrical dimethyl-substituted disulfide bond, was substantially more stable in vivo (t1/2 beta = 88.3 h) than the corresponding IT2, characterized by a disulfide-protected monomethyl substituent bond (t1/2 beta = 60.2 h) compared to the unhindered conjugate IT1 (t1/2 beta = 27.9 h). This family of cross-linking reagents therefore offers advantages, such as minimal perturbation of the protein structure and controlled reactivity due to the thioimidate moiety, as well as the capacity to yield immunotoxins possessing substantial stability in vivo.

Animals↗
PubMed1995

[Stability of 5-fluorouracil solutions according to different parameters].

To follow therapeutic evolutions and to ensure an international homogeneity, "Produits Roche" Laboratories recently modified the conditioning of 5-fluorouracil (5-FU): the Tris buffer was replaced by sodium hydroxide (pH 9.4). For these newly-formulated 5-FU solutions, we studied the optimal conditions of storage according to concentration (0.2; 1.5 and 10 mg/ml), temperature (4, 21 and 37 degrees C), light (without, natural, artificial) and duration (7, 15 and 30 days) for plastic bags, syringes and cassettes. 5-FU was dosaged by High Performance Liquid Chromatography (HPLC). No staining was observed, but for the samples placed at 4 degrees C, we detected macroscopically the apparition of crystals, more or less rapidly according to 5-FU concentration. For plastic bags at 0.2 mg/ml of 5-FU in NaCl (0.9%), 48 hours were sufficient for crystallisation. For preparations at 10 and 50 mg/ml, the formation of crystals occurred in less than 24 hours. However, no microscopic analysis was performed and therefore, in shorter delays, the formation of microcrystals can be envisaged. After 7 days, only the storage in darkness at 21 degrees C yields good results of stability for cassettes and syringes at 50 mg/ml with a degradation of the active component that does not exceed 2%. However, in the same optimal conditions, a storage superior to 14 days cannot be envisaged since we noted a 10% diminution of the active component, then this loss intensifies and reaches 18% after 30 days of storage. For plastic bags at 0.2 and 1.5 mg/ml of 5-FU, we did not measure any significant difference between the various storage conditions, even during 14 days. At 10 mg/ml concentration, a storage during 7 days in darkness can be envisaged with no repercussion on the 5-FU stability. Independently of concentration, special attention must be paid to the risk of crystallisation at 4 degrees C.

Antimetabolites, Antineoplastic↗