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PubMed2004

Rheo-optical properties of silicone oil emulsions in the presence of polymer emulsifiers.

Oil in water emulsions prepared by dispersion of silicone oils into an aqueous solution of hydroxylpropyl methyl cellulose (HPMC) or poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers were characterized by measurements of steady-state shear viscosities, dynamic moduli, and stress-strain sweep curves coupled with optical microscopic observation. The emulsions prepared with HPMC showed solidlike viscoelastic responses and a clear yield stress, whereas the emulsions emulsified by PEO-PPO-PEO block copolymers indicated liquidlike viscoelastic behavior. The difference should be attributed to stronger protective colloidal effects, that is, the former emulsifiers form a more viscoelastic polymer layer adsorbed on the oil droplets than the latter ones. Moreover, the simultaneous optical microscopic observation showed that the emulsions stabilized by HPMC do not flow below the yield stress and beyond the yield stress the movements of oil droplets occur first.

Journal Article↗
PubMed2001

Niacin, poly(ADP-ribose) polymerase-1 and genomic stability.

Nicotinic acid (NA) and nicotinamide (NAM), commonly called niacin, are the dietary precursors for NAD(+) (nicotinamide adenine dinucleotide), which is required for DNA synthesis, as well as for the activity of the enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) for which NAD(+) is the sole substrate. The enzyme PARP-1 is highly activated by DNA strand breaks during the cellular genotoxic stress response, is involved in base excision repair, plays a role in p53 expression and activation, and hence, is thought to be important for genomic stability. In this review, first the absorption, metabolism of niacin to NAD(+), as well as the assessment of niacin status are discussed. Since NAD(+) is important for PARP-1 activity, various aspects of PARP-1 in relation to DNA synthesis and repair, and regulation of gene expression are addressed. This is followed by a discussion on interactions between dietary methyl donor deficiency, niacin status, PARP-1 activity and genomic stability. In vitro studies show that PARP-1 function is impaired and genomic stability decreased when cells are either depleted from NAD(+) or incubated with high concentrations of NAM which is a PARP-1 inhibitor. In vitro as well as animal studies indicate that niacin deficiency increases genomic instability especially in combination with genotoxic and oxidative stress. Niacin deficiency may also increase the risk for certain tumors. Preliminary data suggest that niacin supplementation may protect against UV-induced tumors of the skin in mice, but data on similar preventive effects in humans are not available. NAM has been shown in vitro to have an antioxidant activity comparable to that of ascorbic acid. Data on niacin status and genomic stability in vivo in humans are limited and yield ambiguous results. Therefore, no firm conclusions with respect to optimal niacin intake are possible. As a consequence of oral niacin supplementation, however, NAM levels in the body may increase, which may result in inhibition of PARP-1 and increased genomic instability. More studies are needed to define an optimal level of niacin nutriture in relation to genomic stability and tumorigenesis.

Animals↗
PubMed2000

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties.

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.

ATP-Binding Cassette Transporters↗
PubMed2006

Comparison of the lipase activity in hydrolysis and acyl transfer reactions of two latex plant extracts from babaco (Vasconcellea x Heilbornii Cv.) and Plumeria rubra: Effect of the Aqueous microenvironment.

The enzymatic properties of Plumeria rubra latex have been evaluated for the first time, showing a high activity in both hydrolysis and synthesis reactions, and compared to the biocatalytic behavior of babaco (Vasconcellea x Heilbornii cv.) latex. Both biocatalysts have been optimized by studying the various parameters that influence reaction kinetics. The optimum temperatures for hydrolysis reactions were 50 and 55 degrees C for babaco and Plumeria, respectively. The optimum pH for babaco latex was 7, whereas for Plumeria latex, two optimal pH values (4 and 7) were observed. With regard to esterification and acyl transfer reactions such as alcoholysis and interesterification, the influence of thermodynamic water activity on reaction yields was determined and correlated with water sorption and desorption isotherms. When babaco latex is used as a biocatalyst, optimal synthesis reaction yields are obtained when the enzymatic extract is stabilized at a water activity value of 0.38, which corresponds to a water content of 5.7%. This optimal level of hydration is located on the linear portion of the biocatalyst's sorption isotherm, where the water molecules exhibit high-energy interactions with the protein network. In synthesis reactions (esterification, alcoholysis, and interesterification) biocatalyzed by Plumeria latex, correlation between best reaction yields and water activity cannot be done. Indeed, the sorption isotherm plot has an atypical shape, indicating that water might be trapped in the latex matrix and, consequently, that the water content of the biocatalyst is highly dependent on the hydration history of the latex.

Acylation↗
PubMed2005

Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels.

Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

Escherichia coli Proteins↗
PubMed1993

Complexes of the polyamines spermine, spermidine and putrescine with alpha-lactalbumins.

The effects of polyamines on the spectral properties and thermal stability of different alpha-lactalbumins were measured. Addition of millimolar concentrations of spermine to the Ca(2+)-free (apo) form of bovine or goat alpha-lactalbumin resulted in spectral shifts, in both the far- and near-ultraviolet ranges, similar to those induced by Ca2+ binding. Fluorescence emission spectra of tryptophan residues underwent a pronounced blue shift, concomitant with a decrease in quantum yield. Also, in the presence of spermine, thermal stability was increased. In contrast, in the case of human and to some extent also equine alpha-lactalbumin, the shifts of the CD and fluorescence spectra and the thermal denaturation curves were in the opposite direction. Direct competition of spermine with Mn2+ could be observed for binding to the Ca2+ site of bovine alpha-lactalbumin. As the main binding mode of spermine showed a 1:1 stoichiometry [K1 = (2 +/- 0.5) x 10(4) M-1], we have assumed that binding occurs primarily at this site with one of the distal ammonium groups. In order to accommodate the remaining positively charged ammonium groups of spermine, a binding model was constructed by computer modeling, based on the atomic coordinates of both interacting species. This model makes use of the cluster of negatively charged glutamate residues present in the N-terminal sequence of bovine alpha-lactalbumin, sterically close to the Ca2+ site. The spermine molecule could be nicely fitted in a space delineated by the Ca2+ site, on the one hand, and Glu1, Glu7 and Glu11, on the other. Our model, which has been extended to the shorter polyamines spermidine and putrescine, is able to fully account for the observed stabilization of bovine (and goat) alpha-lactalbumins against thermal denaturation, and for the observed CD and fluorescence spectral shifts. It also explains why human (and equine) alpha-lactalbumin behave differently.

Amino Acid Sequence↗
PubMed1989

Centrifugation of very freshly donated blood may yield platelets unstable to storage in the new generation of containers.

The stability of platelets stored in the second generation of container has been examined further. Platelet concentrates prepared in CPD anticoagulant were found to deteriorate fairly rapidly during storage if prepared from very freshly donated blood, whereas a more than 4-hour delay between donation and platelet isolation yielded platelet concentrates much more stable to storage. Delays of more than 2 h (relative to the time of donation) in the isolation of platelets led to decreases in the aggregation response to low or moderate doses of collagen. Platelets from blood collected into CPD adenine showed decreased aggregation responses relative to equivalent cells from blood collected into adenine-free anticoagulant. It seems that a decreased aggregation response is associated with improved storage properties.

Adenine↗
PubMed1976

Evaluation of labeling procedures and in vivo stability of 99mTc-red blood cells.

Stable labeling of red blood cells (RBCs) with 99mTc, in yields of about 66%, was achieved by using stannous chloride as a reducing agent. In a study of 12 patients, the stability of technetium-labeled RBCs in the circulation varied significantly among individuals, but the average biologic half-life of the labeled cells was 16.8 hr. Although the labeling yield was lower when the labeling was carried out without EDTA with the addition of stannous chloride after the pertechnetate, this sequence appears to be the best for labeling RBCs for use in obtaining images of the blood pool or the cardiovascular system because of the increased viability of the RBCs.

Erythrocytes↗
PubMedDate not supplied

A study of factors affecting the labelling of tartrate with 188Re and the transchelation of the 188Re from the tartrate to a protein.

The formation of 188Re-tartrate for use in transchelation reactions and the transchelation of the 188Re onto albumin was studied. Two labelled tartrate products were separated using a non-traditional mobile phase on ITLC strips. Tartrate labelling yield increases with pH but so does the instability when the product is exposed to air. Lower pH's are preferred when oxygen-free labelling conditions can be achieved. Higher tin levels protect against air oxidation. Stability of the Re-tartrate complex is supported by addition of ascorbic acid and ferrous sulphate, however both these agents decreased the rate of the formation of the Re-tartrate complex. The labelling efficiency of a perrhenate solution decreased with the time for which it is stored prior to the labelling reaction, depending on the radioactive concentration.Re-albumin transchelation efficiency increases with the tartrate concentration, while increased stability of the Re-tartrate complex lowers the transchelation yields of Re-albumin.

Chelating Agents↗
PubMed2006

Colloidal HgTe nanocrystals with widely tunable narrow band gap energies: from telecommunications to molecular vibrations.

A convenient, aqueous-based synthesis of stable HgTe nanocrystals with widely size-tunable room temperature emission between wavelengths of 1.2 to 3.7 mum is demonstrated. By the choice of the thiols, applied as stabilizers, we optimized the growth dynamics, the luminescence quantum yields (up to 40%), and a ligand-exchange procedure, required to transfer the nanocrystals from water to nonpolar organic solvents. The latter is greatly improved and facilitated by the use of mercaptoethylamine as initial stabilizer. The possibility to tune the HgTe nanocrystal sizes from 3 to 12 nm and to control their surface functionalities (hydrophobic and hydrophilic) makes them very promising for the development of infrared optical devices, emitting in the wavelength region between the telecommunications and the molecular vibrations.

Journal Article↗
PubMed2005

Characterization of adducts formed in the reaction of 2-chloro-4-methylthiobutanoic acid with 2'-deoxyguanosine.

2-chloro-4-methylthiobutanoic acid (CMBA) is a direct-acting mutagen found in salt-nitrite-treated Sanma fish or similarly treated methionine solution. In this study, CMBA was reacted with 2'-deoxyguanosine (dG) in phosphate buffer (pH 7.4) at 37 degrees C. The HPLC-UV analysis showed that two products were mainly formed during the reaction. These were isolated, purified by semipreparative HPLC, and characterized as N7-guanine adducts: N7-(3-carboxy-3-methylthiopropyl)guanine (A1) and N7-(1-carboxy-3-methylthiopropyl)guanine (A2). Furthermore, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis was employed to investigate the possible formation of minor products during the time-course of the reaction of CMBA with dG. It was found that N7-dG adducts, the precursors of A1 and A2, were formed early in the reaction and that subsequently the spontaneous depurination occurred to yield stable N7-guanine adducts A1 and A2. Stability studies in phosphate buffer (pH 7.4) at 37 degrees C showed that the amount of each N7-dG adduct decreased rapidly with a half-life of 6 h and 4 h to yield A1/A2, respectively. A regioisomer of N7-dG adducts was also observed in the LC/ESI-MS/MS analysis, but it was not characterized in detail because it was present only in trace amounts. On the basis of structural features, A1 and A2 seemed to be formed from the reaction of dG with 1-methyl-2-thietaniumcarboxylic acid, an intermediate resulting from the cyclization of CMBA. However, A2 might also have formed from the direct reaction of dG and CMBA. N7-Alkylation of the guanine residue and subsequent depurination are known to produce apurinic sites in DNA that induce point mutations and may be responsible for the observed CMBA-induced mutagenesis.

Butyrates↗
PubMed1985

The androgen receptor in normal human foreskin. I. Stabilization and identification of two receptor subunits.

Use of glycerol and the protease inhibitor phenylmethylsulfonyl fluoride resulted in a fourfold increase in the yield of androgen receptor from human neonatal foreskin and a tenfold increase in stability of this receptor. The general physical properties of the androgen-binding component isolated from the cytosol of human neonatal foreskin were found to be consistent with those previously reported for androgen receptors. The synthetic androgen methyltrienolone (R1881) was bound with high affinity (a mean dissociation constant of 0.51 nmol/L) and low capacity (5.6 fmol/mg of protein); maximum binding required 4 hours. Affinity of the receptor was highest for R1881 followed by dihydrotestosterone, testosterone, and progesterone. 17 beta-Estradiol showed little or no competition. The receptor exhibited a change of sedimentation coefficient from 8S to 4S upon treatment with 0.1 mol/L potassium chloride. Isoelectric focusing demonstrated the existence of two molecular species with apparent isoelectric points of 7.2 and 5.7.

Binding, Competitive↗
PubMed2006

Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase.

Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.

Alkaline Phosphatase↗
PubMed2001

Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone-1-butanol-ethanol solvent by Clostridium acetobutylicum.

A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch by Clostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent, i.e. 1.5-2 times more than with pure maltose or glucose used as carbon sources. Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch. The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation. The pH optima for alpha-amylase and glucoamylase were found to be at 5.3 and 4.0-4.4, respectively. alpha-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0-8.0 after a 1-d incubation at 37 degrees C. Since C. acetobutylicum alpha-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch. However, the C. acetobutylicum glucoamylase was only stable at pH 4-5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37 degrees C.

1-Butanol↗
PubMed1995

Synthesis and properties of a Hoechst-like minor-groove binding agent tethered to an oligodeoxynucleotide.

A small Hoechst-like DNA groove-binding fluorophore carrying a terminal bromoacetimido linker has been synthesized. Individual diastereomeric oligodeoxynucleotide dodecamers containing a thiol-based linker attached to an internucleotide phosphoramidate within a sequence of dA-dT residues were each covalently labeled with the new fluorophore. One isomer of the DNA-fluorophore conjugate (Isomer B) hybridizes to the complementary single-stranded target DNA and the presence of the tethered fluorophore results in both increased duplex stability and an enhanced fluorescent signal, presumably due to the fluorophore binding in the dA-dT rich minor groove. Duplex stability is increased by 2-3 degrees C and the fluorescence quantum yield for the fluorophore increases four-fold. In contrast, the other diastereomer (Isomer A) exhibits reduced helix stability (approximately 2 degrees C) and only slight changes in fluorescence intensity upon hybridization.

Base Sequence↗
PubMed2005

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose.

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.

Aldose-Ketose Isomerases↗
PubMed2003

Conferring thermostability to mesophilic proteins through optimized electrostatic surfaces.

Recently, there have been several experimental reports of proteins displaying appreciable stability gains through mutation of one or two amino acid residues. Here, we employ a simple theoretical model to quickly screen mutant structures for increased thermostability through optimization of the protein's electrostatic surface. Our results are able to reproduce the experimental observation that elimination of like-charge repulsions and creation of opposite-charge attractions on the protein surface is an efficient method to confer thermostability to a mesophilic protein. Using Poisson-Boltzmann electrostatics, we calculate relative protein stabilities for the exhaustive surface mutagenesis of the cold shock, RNase T1, and CheY proteins. Comparison with 25 experimentally characterized cold shock protein mutants reveals an average correlation of 0.86. The model is also quantitatively accurate when reproducing the experimental D49A and D49H mutant stabilities of RNase T1. This work represents the first comprehensive in silico screening of mutant candidates likely to confer thermostability to mesophilic proteins through optimization of surface electrostatics. Systematic single mutant, followed by double mutant, screening yields a limited number of mutant structures displaying significant stability gains suitable for subsequent experimental characterization.

Amino Acid Sequence↗
PubMed2002

Stabilization of a terminal inversion duplication of 8p by telomere capture from 18q.

Terminal inversion duplications of the short arm of chromosome 8 are one of the more common chromosome rearrangements in humans. We report an infant with multiple congenital anomalies, in whom karyotype analysis showed a terminal inversion duplication of 8p including additional material at the distal end of the derivative chromosome, shown to be of chromosome 18q origin. Terminal inversion duplications of 8p are the result of meiotic recombination between inverted olfactory gene receptor repeats in 8p. This recombination generates a dicentric intermediate that breaks during anaphase, and the broken chromosome end is stabilized by telomere healing or telomere capture. The origin of the telomeric region in the majority of constitutional chromosome deletions studied to date was shown to be from telomere healing; the de novo addition of telomeric repeats. In the proband a cytogenetically detectable piece of chromosome 18q was present on the distal end of the derivative 8, suggesting that this chromosome was stabilized by telomere capture of 18q. FISH analyses of additional cases may yield information as to whether telomere capture or telomere-healing events are the predominant mechanism of chromosome stabilization in terminal inversion duplications of 8p.

Adult↗