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PubMed2005

Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state.

Phosphotriesterase from Pseudomonas diminuta (PTE) is an extremely efficient metalloenzyme that hydrolyses a variety of compounds including organophosphorus nerve agents. Study of PTE has been hampered by difficulties with efficient expression of the recombinant form of this highly interesting and potentially useful enzyme. We identified a low-level esterolytic activity of PTE and then screened PTE gene libraries for improvements in 2-naphthyl acetate hydrolysis. However, the attempt to evolve this promiscuous esterase activity led to a variant (S5) containing three point mutations that resulted in a 20-fold increase in functional expression. Interestingly, the zinc holoenzyme form of S5 appears to be more sensitive than wild-type PTE to both thermal denaturation and addition of metal chelators. Higher functional expression of the S5 variant seems to lie in a higher stability of the metal-free apoenzyme. The results obtained in this work point out another-and often overlooked-possible determinant of protein expression and purification yields, i.e. the stability of intermediates during protein folding and processing.

Chelating Agents↗
PubMed1977

[Comparative studies on the breaking strength of tissue adhesives of the butylcyanoacrylate type].

Four tissue adhesives of the butylcyano-acrylate type were tested for breaking strength. The addition of dyes and stabilizers results in an increase in breaking strength. Adhesives coloured with an anthraquinone dye and stabilized with SO2 significantly surpass in breaking strength adhesives that are only coloured or stabilized. Fiomed II (coloured, stabilized with SO2) yields significantly (at P=0.001) better values than Histoacryl blue. Addition of the dye alone seems to exert a better effect on breaking strength than addition of the stabilizer alone. As evidenced by our results, the breaking strength is in the order: 1. Fiomed II (coloured, stabilized with SO2), 2. Histoacryl blue, 3. Fiomed III (coloured, without SO2), 4. Fiomed I (without dye, stabilized with SO2).

Adult↗
PubMed1995

Stability and ear advantage for dichotically presented environmental sound stimuli: development during early childhood.

55 right-handed children with no family history of left-handedness received two dichotic presentations of environmental sounds. The mean test-retest interval was 9 days. Raw accuracy scores yielded acceptable temporal stability (rs > .72). The expected consistent left-ear advantage for environmental sound stimuli was only evident in younger and less mature children. Five- and 6-yr.-old children, particularly those with strong right-hand preferences, had a right-ear advantage for the stimuli. Their computed laterality coefficients showed low and nonsignificant test-retest reliabilities, however. Three- and 4-yr.-old children, particularly those with weak hand preferences, had a left-ear advantage for the same stimuli. Of the 3- and 4-yr.-old children who did not have strong right-hand preferences, 92% demonstrated consistent ear advantages across testing sessions and their laterality coefficient test-retest correlations were significant. In contrast, only 47% of the strongly right-handed 3- and 4-yr.-old children and 71% of the strongly right-handed 5- and 6-yr.-old children had consistent ear advantages for the same stimuli. It is suggested that the 3- and 4-yr.-old children processed these stimuli according to endogenous, stimulus-specific brain mechanisms and that learned processing strategies overrode these mechanisms for the 5- and 6-yr.-old children.

Attention↗
PubMed1999

Heat stability of the potato tuber ADP-glucose pyrophosphorylase: role of Cys residue 12 in the small subunit.

Most of the ADP-glucose pyrophosphorylases from different sources are stable to a heat treatment. We found that in the potato (Solanum tuberosum L.) tuber enzyme, the intermolecular disulfide bridge located between Cys12 of the small subunits is responsible for the stability at 60 degrees C. When this unique disulfide bond is cleaved the enzyme is stable up to 40 degrees C. Mutation of Cys12 in the small subunit into either Ala or Ser yielded enzymes with stability similar to the reduced form of the wild type. Concurrently, the enzyme with a truncated small subunit on the N-terminal was stable only up to 40 degrees C. Thus, the N-terminal is important for the stability of the enzyme because of the presence of a disulfide bond.

Adenosine Diphosphate Glucose↗
PubMed1994

Thermostabilized chemical derivatives of horseradish peroxidase.

Horseradish peroxidase finds a variety of uses in analysis, immunology, organic synthesis, and biosensors. Although moderately stable, its applicability to biosensors and other fields would be greatly enhanced if it could be made yet more stable. Appropriate chemical modification can substantially stabilize enzymes. Here we describe the use of bis-imidates and of bis-succinimides to modify free amino groups of commercial horseradish peroxidase under mild conditions of pH and temperature. Imidates yielded a marginal stabilization. Some of the succinimide derivatives, however, are much more thermostable than the native enzyme. Apparent half-lives indicate stabilizations of 6- to 23-fold, depending on the bis-succinimide used. These modifications preserve the carbohydrate side chains for subsequent reaction or immobilization.

Biosensing Techniques↗
PubMed2003

Multicolor luminescence patterning by photoactivation of semiconductor nanoparticle films.

Indiscriminate adsorption of nanoparticles (NPs) significantly complicates the preparation of mesoscale NP patterns considered as enabling technology for many devices and processes. Instead of selected chemical functionalization of the substrate surface prior to the assembly of nanocolloids, the required optical properties - in our case, high quantum yield luminescence - are imparted to the layer-by-layer assembled films by spatially selected photoactivation. The films are made by sequential adsorption of a positively charged polyelectrolyte and a negatively charged CdSe/CdS aqueous dispersion with an initial quantum yield of 0.5-2%. The photoactivation process takes place in the presence of oxygen and may be accompanied by photoetching. A 50-500-fold increase in the luminescence intensity of CdSe/CdS citrate-stabilized particles (quantum yield 25-45%) after visible light illumination provides excellent pattern contrast. Micron scale luminescence patterns were produced from NPs of various CdSe core diameters with red, yellow, and green emission. It was also demonstrated that different emission colors such as orange and green can be combined in one image by taking advantage of spatially selective photoetching. The presented optical patterning technique significantly simplifies the preparation of luminescence patterns as compared to conventional methods. The high signal-to-noise ratio associated with it is essential for optical devices, information processing, and biophotonics. The most immediate use of this approach is expected in cryptography and cell monitoring.

Cadmium Compounds↗
PubMed1997

Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.

Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation.

Apoproteins↗
PubMed2001

Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily.

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures.

Carrier Proteins↗
PubMed1981

The geometry of diarthrodial joints, its physiologic maintenance, and the possible significance of age-related changes in geometry-to-load distribution and the development of osteoarthritis.

Diarthrodial joints are governed by physiologic mechanisms that maintain stability and an equitable distribution of load. Modeling continues throughout life to maintain the necessary physiologic incongruity. However, in old age the system seems to go awry, and the result is an increasing congruity yielding possibly increased stability but interfering with cartilage nutrition and altering the distribution of load. The increasing maldistribution of load, with age, it is proposed, mechanically overtaxes the previously underloaded and, presumably, atrophic cartilage. Overtaxing the cartilage in turn leads to further depletion of proteoglycans, collagen fraying and eventually osteoarthritis. Thus arthritis, at least in one of its forms, appears to be inevitable because of the maldistribution of load that results from the age-related changes in joint shape possibly dictated by the joint's requirement for stability.

Aging↗
PubMed1996

[Purification of EcoO44I restriction endonuclease in Escherichia coli O44 isolated from an affected human].

A restriction endonuclases (ENase) designated EcoO44I was purified without non specific nucleases from enteropathogenic Escherichia coli O44 Hiromi strain of affected human origin. The yield was 1, 100 units/g of wet cells. The EcoO44I ENase recognized and cleaved the specific sequence of 5'-GGTCTC-3' (1/5) as was the case with Eco31I or BsaI ENase. Because of the stability and high yield, EcoO44I would be useful for recombinant DNA technology after isolation of EcoO44-positive, avirulent mutant strains of E. coli O44 Hiromi.

DNA Restriction Enzymes↗
PubMed2004

Preclinical characterisation of 111In-DTPA-trastuzumab.

Trastuzumab (Herceptin) is a recombinant humanised IgG1 monoclonal antibody against the human epidermal growth factor receptor 2 (HER2), used for metastatic breast cancer treatment. Radiolabelled trastuzumab may have several future applications for diagnostic use. The aim of the present study was to develop clinical grade (111)Indium ((111)In) radiolabelled trastuzumab, to evaluate the stability and immunoreactivity of the tracer and to perform a biodistribution study in human tumour-bearing mice. Trastuzumab was radiolabelled with (111)In using DTPA as a chelator. (111)In-DTPA-trastuzumab (labelling yield 92.3+/-2.3%, radiochemical purity 97.0+/-1.5%) is stable in PBS when stored at 4 degrees C for more than 14 days. The immunoreactive fraction determined by cell-binding assays, using the HER2-overexpressing human ovarian SK-OV-3 tumour cell line, was 0.87+/-0.06. Biodistribution and tumour targeting were studied in HER2 receptor-positive and -negative tumour-bearing athymic mice. The HER2-positive tumour showed (9.77+/-1.14% injected dose per gram (ID g(-1))) substantial uptake of the labelled antibody already after 5 h. The difference in uptake between HER2-positive versus -negative tumours was even more pronounced 3 days after injection (16.30+/-0.64% ID g(-1)), and was visualised by radioimmunoscintigraphy. Liver, spleen and kidney showed marked tracer uptake. In summary, trastuzumab can be efficiently radiolabelled with (111)In with high labelling yields and high stability. (111)In-DTPA-trastuzumab selectively binds to the human HER2 receptor both in vitro and in vivo in animals. Therefore, (111)In-DTPA-trastuzumab appears suitable for clinical use.

Animals↗
PubMed2005

Biomechanical testing of the lumbar facet interference screw.

STUDY DESIGN: An in vitro study was conducted to determine the biomechanical properties of a new simple, percutaneous, posterior fixation technique for the lumbar spine involving a new implant, the so-called Lumbar Facet Interference Screw. OBJECTIVES: The purpose of this study was to compare the biomechanical properties of this new fixation device with translaminar and pedicle screw fixation. SUMMARY OF BACKGROUND DATA: Several techniques were described to perform a minimal invasive posterior stabilization of the lumbar spine after an anterior lumbar interbody fusion procedure. Yet, due to the high complexity of these minimally invasive surgical procedures, currently, hardly any of these percutaneous posterior fixation techniques is carried out routinely. METHODS: Ten human lumbar spines were tested in flexion, extension, axial rotation, and lateral bending using a nonconstrained testing method. First, all motion segments were evaluated intact (group 1). After complete discectomy of L4-L5, the following stabilization techniques were tested sequentially (n = 10/group): group 2: "stand-alone" cage; group 3: cage plus translaminar screws; group 4: cage plus Lumbar Facet Interference Screw; and group 5: cage plus pedicle screws. Stiffness, ranges of motion, and neutral and elastic zones were determined. RESULTS: In comparison to the intact motion segment, the "stand-alone" cage showed a significantly higher (P < 0.05) range of motion, neutral zone, and elastic zone and a significantly lower (P < 0.05) stiffness in extension and rotation. Generally, all fixation techniques using cages plus posterior stabilization decreased range of motion, neutral zone, and elastic zone and increased stiffness in comparison to the "stand-alone" cage group. There was no significant difference between the cage plus interference screw and the cage plus translaminar screw group in all test modes. In comparison to the 2 facet joint stabilization techniques, pedicle screw stabilization decreased (P < 0.01) range of motion, neutral zone, and elastic zone and increased (P < 0.01) stiffness significantly in flexion and rotation. CONCLUSIONS: Results of this study indicate that the new Lumbar Facet Interference Screw fixation yields initial biomechanical stability similar to translaminar screw fixation, yet inferior biomechanical stability compared to pedicle screw fixation. Although these results are encouraging, additional biomechanical studies including cyclic loading tests have to evaluate the mid- and long-term stabilization capacity of this new minimally invasive fixation technique before human application.

Adult↗
PubMed2003

Rx for low cash yields.

Certain strategies can offer not-for-profit hospitals potentially greater investment yields while maintaining stability and principal safety. Treasury inflation-indexed securities can offer good returns, low volatility, and inflation protection. "Enhanced cash" strategies offer liquidity and help to preserve capital. Stable value "wrappers" allow hospitals to pursue higher-yielding fixed-income securities without an increase in volatility.

Financial Management, Hospital↗
PubMed1994

Denaturation of free and complexed bovine trypsinogen with the calcium ion, dipeptide Ile-Val and basic pancreatic trypsin inhibitor (Kunitz).

Thermal and chemical denaturation has been used to probe changes in the thermodynamic stability of trypsinogen upon complexation with calcium ion and with ligands, which induce the conformational transition of the zymogen to the trypsin-like form. Chemical and thermal unfolding curves of ligand-free trypsinogen at pH 5.8 are cooperative and yielded the following stability parameters: the free energy change of denaturation delta Gden = 44.8 kJ/mol, the denaturation temperature = 65.7 degrees C, the enthalpy change of denaturation delta Hden at the denaturation temperature Tden = 607 kJ/mol and the heat capacity change of denaturation delta Cp,den = 12.4 kJ.mol-1.K-1. Fast phases of both unfolding and refolding of trypsinogen proceed on a time scale of seconds and fit to a single exponential. At pH 5.8, the calcium ion increases the conformational stability delta Gden by 7.1 kJ/mol, Tden by 2.6 K and delta Hden by 80 kJ/mol, but does not induce any substantial structural change in the trypsinogen molecule, as revealed by 1H-NMR spectra. The trypsin-like form of trypsinogen, generated by complexation of the zymogen with the dipeptide Ile-Val and/or basic pancreatic trypsin inhibitor (Kunitz), is characterized by increase of delta Hden by 134 kJ/mol and Tden by 2.5 K, which may be attributed to the additional energy required to disrupt the rigidified activation domain in the complexed trypsinogen.

Animals↗
PubMed2004

[Body condition and metabolic stability as the basis for high milk yield and undisturbed fertility in dairy cows--a contribution for deduction of reference values].

The target of this study was to describe the interactions between body condition and various descriptors of yield and fertility. It was aimed to identify an optimal conditional range to be used in herd management which combines high milk yield with acceptable fertility traits and general health. For this purpose, backfat thickness was measured by ultrasound at 46111 dairy cows on 78 different farms and was subsequently related to production variables. Negative energy balance is getting more intense and prolonged with increasing milk yield. However a conditional nadir below 10 mm leads to decreased milk production. To reach a high production level without an increasing incidence of health disorders, conditional nadir should not decline below 13 mm backfat thickness on herd average. Lower value only lead to negligibly higher milk yield but cause a distinctively higher risk of fertility problems and culling. High herd yields do not have to be at expense of reproduction performance and can be achieved without extreme body condition losses. An efficient herd management can offset depression in fertility, which commonly is combined with increasing milk yield. A standard curve for backfat thickness throughout lactation is suggested to be used in dairy herd management.

Adipose Tissue↗
PubMed2005

Effect of axial ligation or pi-pi-type interactions on photochemical charge stabilization in "two-point" bound supramolecular porphyrin-fullerene conjugates.

Two types of structurally well-defined, self-assembled zinc porphyrin-fullerene conjugates were formed by "two-point" binding strategies to probe the effect of axial ligation or pi-pi-type interactions on the photochemical charge stabilization in the supramolecular dyads. To achieve this, meso-tetraphenylporphyrin was functionalized to possess one or four [18]crown-6 moieties at different locations on the porphyrin macrocycle while fullerene was functionalized to possess an alkyl ammonium cation, and a pyridine or phenyl entities. As a result of the crown ether-ammonium cation complexation, and zinc-pyridine coordination or pi-pi-type interactions, stable zinc porphyrin-fullerene conjugates with defined distance and orientation were formed. Evidence for the zinc-pyridine complexation or pi-pi-type interactions was obtained from the spectral and computational studies. Steady-state and time-resolved emission studies revealed efficient quenching of the zinc-porphyrin singlet excited state in these dyads, and the measured rates of charge separation, k(CS) were found to be slightly better in the case of the dyads held by axial coordination and crown ether-cation complexation. Nanosecond transient absorption studies provided evidence for the electron transfer reactions, and these studies also revealed charge stabilization in these dyads. The lifetimes of the radical ion pairs were found to depend upon the type of porphyrins utilized to form the dyads, that is, porphyrin possessing the crown ether moiety at the ortho position of one of the phenyl rings yielded prolonged charge stabilized states. Addition of pyridine to the supramolecular dyads eliminated the zinc-pyridine coordination or pi-pi-type interactions of the "two-point" bound systems due to the formation of a new zinc-pyridine axial bond thus giving a unique opportunity to probe the effect of axial coordination or pi-pi interactions on k(CS) and k(CR). Under these conditions, the measured electron transfer rates revealed faster k(CS) and slower k(CR) as compared to those obtained in the absence of added pyridine. The evaluated lifetimes of the radical ion-pairs were found to be hundreds of nanoseconds and were longer in the presence of pyridine.

Electrochemistry↗
PubMed1989

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid.

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

Animals↗
PubMed2026

Ethanol pretreatment drives microbial community adaptation to overcome acidification in high-solid anaerobic digestion of food waste under rapid organic loading shock.

This study investigated how ethanol pretreatment (EP) enhances the resilience of high-solid anaerobic digestion against rapid organic loading shocks. Semi-continuous reactors fed with either untreated or EP-treated food waste were compared, and the underlying mechanisms were elucidated by integrating thermodynamic calculations with metagenomic analyses. At an organic loading rate of 6.0&#xa0;g VS/(L&#x2d1;d), the control group (untreated) collapsed due to the accumulation of propionate and other longer-chain volatile fatty acids (VFAs), resulting in a methane yield decrease exceeding 70%. In contrast, the EP group maintained stability, exhibiting a methane yield decrease of less than 5%, with VFAs dominated by readily degradable acetate. Thermodynamic analysis confirmed that EP significantly lowered the energy barriers for VFA degradation. Metagenomic analysis revealed that both propionate/butyrate activation pathways (with lower energy cost or independence from acetyl-CoA) and syntrophic acetate oxidation were activated in the EP group, thereby avoiding the VFA metabolic stress observed in the control group. Furthermore, higher abundances of conductive type IV pili genes, Complex II, and archaeal V/A-type ATPase were detected in the EP group, suggesting the establishment of direct interspecies electron transfer and enhanced electron flux and energy capture efficiency. Moreover, under high loading conditions, only a few high-abundance metagenome-assembled genomes (MAGs) were detected in the control group, while multiple MAGs carrying identical VFA-degrading enzyme systems were identified in the EP group. The functionally redundant microbiota, unobstructed VFA metabolic pathways, and efficient electron transfer and energy supply collectively sustained the stability of the EP group under loading shocks.

Anaerobic digestion↗