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PubMed1983

Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties.

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.

Amino Acids↗
PubMedDate not supplied

Brighter near-field optical probes by means of improving the optical destruction threshold.

The optical destruction thresholds of conventionally etched and tube-etched near-field optical probes were measured. One of the main advantages of tube-etched tips is their smooth glass surface after taper formation. Presumably for this reason, a destruction limit of over 120 microJ was obtained, almost twice as large as that of the rougher, conventionally etched fibre probes. The use of additional adhesion layers (Ti. Cr, Co and Ni) between the glass surface and the aluminium coating produced, especially for tube-etched tips, a significant increase in the optical destruction threshold. With increasingly thin metal coatings, the use of a protection coating that prevents corrosion during aging is recommended. An additional increase in optical stability was achieved by applying mixed-metal coatings: alternating thin titanium and thick aluminium layers yielded fibre probes with superior properties that achieved average optical destruction thresholds of > 270 microJ. This is an increase in stability of > 400% compared with conventionally fabricated near-field optical tips.

Journal Article↗
PubMed1981

Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids.

Ten plasmids were isolated as covalently closed circular deoxyribonucleic acid from antibiotic-resistant thermophilic bacteria. Of the 10 plasmids tested, 2 could transform Bacillus subtilis, yielding resistance to specific antibiotics. Plasmid pTB20 (2.8 X 10(6) daltons, approximately 24 copies per chromosome) specifies resistance to tetracycline (Tcr), whereas pTB19 (17.2 X 10(6) daltons, approximately 1 copy per chromosome) renders the host resistant to both kanamycin and tetracycline (KMrTcr). Three plasmids were not self-transmissible. The restriction endonuclease cleavage maps of the two plasmids, pTB19 and pTB20, were constructed. pTB19 and pTB20, both of which were originally isolated from thermophilic bacilli, were tested for stability in B. subtilis. Digestion of pTB19 followed by ligation yielded deletion plasmids pTB512 (Kmr), pTB52 (Tcr), and pTB53 (KmrTcr). Determinants of Kmr, Tcr, and DNA replication were associated with EcoRI fragments R1b (4.2 X 10(6) daltons), R3 (2.8 X 10(6) daltons), and R1a (4.2 X 10(6) daltons), respectively. Restriction endonuclease cleavage maps of pTB51, pTB52, and pTB53 were constructed. Tetracycline resistance of pTB20 was confirmed to be in the EcoRI fragment (1.85 X 10(6) daltons).

Bacillus subtilis↗
PubMed1995

Characterization of a hepatic protein in nonhuman primates that binds mibolerone but not dihydrotestosterone or methyltrienolone.

During our studies of the hepatic androgen receptor in cynomolgus monkeys, tritiated mibolerone +/- a 200-fold excess of unlabeled mibolerone has been used to determine specific binding in cytosol. During time-course studies, high-capacity, unsaturable binding of [3H]mibolerone was noted after short-term incubations (4 h, 4 degrees C). When hepatic cytosol from male monkeys was incubated for 18 h at 4 degrees C, the high-capacity binding disappeared; saturable, high-affinity binding with characteristics consistent with the androgen receptor then could be identified. The characterization of [3H]mibolerone binding in molybdate-stabilized hepatic cytosol using sucrose density gradients and gel filtration yielded an unstable binding peak in addition to that of the androgen receptor. This lower molecular weight protein identified by gel filtration did not bind other androgens, including methyltrienolone, and did not have characteristics of other binding proteins that have been identified previously. This protein was not precipitated from 30% ammonium sulfate, which allowed it to be separated from the androgen receptor. Binding to this protein in ovariectomized female monkeys did not disappear with extended incubation at 4 degrees C, suggesting greater stability or a higher capacity. The function of this protein is not known, but both triamcinolone acetonide and contraceptive progestins appeared to displace tritiated mibolerone that was bound to it. This high-capacity binding of mibolerone interferes in the assessment of androgen receptor levels in these females unless it is eliminated. The synthetic androgen methyltrienolone does not bind to this protein and is a better choice for defining binding to the androgen receptor in these tissues.

Ammonium Sulfate↗
PubMed2006

Wear resistance and mechanical properties of highly cross-linked, ultrahigh-molecular weight polyethylene doped with vitamin E.

Our hypothesis was that cross-linked, ultrahigh-molecular weight polyethylene (UHMWPE) stabilized with vitamin E (alpha-tocopherol) would be wear-resistant and fatigue-resistant. Acetabular liners were radiation cross-linked, doped with vitamin E, and gamma-sterilized. Hip simulator wear rate of vitamin E-stabilized UHMWPE was approximately 1 and 6 mg/million-cycles in clean serum and in serum with third-body particles, respectively, a 4-fold to 10-fold decrease from that of conventional UHMWPE. The ultimate strength, yield strength, elongation at break, and fatigue resistance of vitamin E-stabilized UHMWPE were significantly higher than that of 100 kGy-irradiated and melted UHMWPE, and were unaffected by accelerated aging. Rim impingement testing with 3.7-mm-thick acetabular liners up to 2 million-cycles showed no significant damage of the cross-linked liners compared with conventional, gamma-sterilized in inert UHMWPE, vitamin E-stabilized liners. The data indicate good in vitro wear properties and improved mechanical and fatigue properties for vitamin E-stabilized, cross-linked UHMWPE.

Fatigue↗
PubMed1994

Hybridoma stability.

Hybridoma stability issues include mutations, chromosome losses, and the potential effects of process variables on the yield, quality and homogeneity of the Monoclonal Antibody (MAb) product. MAb production by murine hybridomas is typically unstable in the early stages after fusion but repeated cloning normally produces stable clones. The stability of hybridomas and the consistency of the MAbs produced during extended high density perfusion cultures at Xoma Corporation were evaluated. Cell stability was assessed by recovering cells from the bioreactors at different intervals and comparing their growth and product formation kinetics and yields to those of cells started fresh from the corresponding Manufacturer's Working Cell Banks. Product consistency was evaluated in the crude harvests and in the corresponding purified MAb lots by biochemical and functionality tests including: SDS-PAGE (reducing and non-reducing), IEF, HPLC (size exclusion and cation exchange), peptide mapping, N-terminal sequencing, carbohydrate composition and binding assays. Several murine hybridomas were studied during runs lasting several months and found to be stable by all criteria employed. Such results support the viability of extended hollow fiber perfusion cultures for reproducible production of murine MAbs. Selecting stable clones and understanding the effects of process variables on the quantity and quality of the MAbs are keys to controlling hybridoma stability during the manufacturing process.

Animals↗
PubMed2005

Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module.

We developed a new fully automated method for the synthesis of [18F]fluoromisonidazole ([18F]FMISO) by modifying a commercial FDG synthesizer and its disposable fluid pathway. A three-step procedure was used to prepare the tosylate precursor, 1-(2'-nitro-1'-imidazolyl)-2-O-tetrahydrofuranyl-3-O-toluenesulfonylpropanediol. Using glycerol as the starting material, the precursor was synthesized with a yield of 21%. The optimal labeling conditions for the automated synthesis of [18F]FMISO was 10 mg of precursor in acetonitrile (2 ml heated at 105 degrees C for 360 s, followed by heating at 75 degrees C for 280 s and hydrolysis with 1 N HCl at 105 degrees C for 300 s. Using 3.7 GBq of [18F]F- as a starting activity, [18F]FMISO was obtained with high end-of-synthesis (EOS) radiochemical yields of 58.5+/-3.5% for 60.0+/-5.2 min with high-performance liquid chromatography (HPLC) purification. When solid-phase purification steps were added, the EOS radiochemical yields were 54.5+/-2.8% (337+/-25 GBq/micromol) for 70.0+/-3.8 min (n=10 for each group, decay-corrected). With a high starting radioactivity of 37.0 GBq, we obtained radiochemical yields of 54.4+/-2.9% and 52.8+/-4.2%, respectively (n=3). The solid-phase purification removed unreacted [18F]fluoride and polar impurities before the HPLC procedure. Long-term tests showed a good stability of 98.2+/-1.5%. This new automated synthesis procedure combines high and reproducible yields with the advantage of using a disposable cassette system.

Drug Evaluation, Preclinical↗
PubMed2001

Calcium alginate gel: a biocompatible and mechanically stable polymer for endovascular embolization.

The development and optimization of calcium alginate for potential use in endovascular occlusion was investigated by testing its in vitro and in vivo mechanical stability and biocompatibility. The compressive resistance, rheology, and polymer yield of reacted alginate, and the polymer viscosity of unreacted alginate, were assessed. Biocompatibility was tested by injecting calcium alginate into the kidney capsule of rats. The reactivity of alginates with various structures and levels of purity were compared visually and histologically. Results suggest that calcium alginate is a biocompatible and mechanically stable gel for endovascular applications. Purified alginates exhibited compressive strength of 22 kPa and above at 40% compression, with no significant loss in elasticity. Purified alginate strength was significantly higher than that of crude alginates (p < 0.08). Purified alginates also exhibited significantly lower tissue reaction than crude alginates (p < 0.05). Of the alginates tested, purified high guluronic acid alginates (PHG) exhibited optimal strength and polymer yield, increased biocompatibility, and decreased viscosity. Clinical embolization treatments may be improved with the development of stable and biocompatible polymers such as calcium alginate. Possible uses of improved endovascular polymers include treating arteriovenous malformations (AVMs), aneurysms, blood flow to tumors, and vascular hemorrhaging.

Alginates↗
PubMed2002

The nature of the indenyl effect.

The eta(5)-to-eta(3) coordination shift of cyclopentadienyl (Cp=C(5)H(5)(-)) and indenyl (Ind=C(9)H(7)(-)) ligands in molybdenocene complexes, [(eta(5)-Cp')(eta(5)-Cp)Mo(CO)(2)](2+) (Cp'=Cp or Ind), driven by a two-electron reduction of those species, was studied and compared by means of molecular orbital calculations (B3LYP HF/DFT hybrid functional, DZP basis sets). The results obtained, in terms of optimized geometries, relative energies, and bond analysis parameters, compare well with the experimental data, and verify the well-known indenyl effect, that is, a significantly more facile eta(5)-to-eta(3) rearrangement for the indenyl ligand when compared to cyclopentadienyl. However, the study of the folding of free Cp and Ind, combined with the (eta(5/3)-Cp')-M bond analysis, shows that the observed difference is not the result of an intrinsic characteristic of the indenyl ligand, such as the traditionally accepted aromaticity gain in the benzene ring formed in eta(3)-Ind complexes. Instead, it is directly related to the Cp'-M bond strength. While the difference in the energy required to fold the two free ligands is negligible (< or =1 kcal mol(-1) for folding angles up to 20 degrees), the (eta(5)-Cp)-M bond is stronger than that of (eta(5)-Ind)-M; however, the opposite situation is found for the eta(3) coordination mode. The net result, for Cp'=Ind, is a destabilization of the eta(5) complexes and a stabilization of the eta(3) intermediates or transition states yielding smaller activation energies and faster reaction rates for processes in which that is the rate-determining step.

Journal Article↗
PubMedDate not supplied

Ultrasensitive plasmid mapping by high performance capillary electrophoresis.

This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely related plasmids with three different restriction enzymes. The plasmids express full length and truncated forms of a growth factor receptor oncogene product and were digested with HpaII, HaeIII and RsaI. The resulting oligonucleotide fragments were under 2000 base pairs in length, a size well suited to separation by HPCE with linear polyacrylamide as a sieving matrix. Plasmid mapping is an essential tool in biotechnology both for the design of an expression system and for monitoring the stability of the expression system during fermentation. HPCE can yield much higher resolution of oligonucleotides than attainable in conventional agarose gel electrophoretic procedures for plasmid mapping. In the examples described here, the HpaII digests provided the surest identification of individual plasmids in the HPCE analysis and could discriminate among all four plasmids. In conventional slab electrophoresis, however, the RsaI digests provided the best discrimination, although two of the plasmids in this system yielded essentially identical electrophoretic patterns. Hence the optimal restriction enzyme for plasmid mapping applications with HPCE may differ from that selected on the basis of conventional slab gel analysis, and the former technique can provide higher discrimination among related plasmids. The advantages of the HPCE format with respect to speed, low sample consumption and resolution are described.

Electrophoresis, Agar Gel↗
PubMed2003

DNA capillary electrophoresis using poly(vinyl alcohol). II. Separation media.

We represent the first extensive study on DNA capillary electrophoresis using various poly(vinyl alcohol) (PVAL)-related polymers as separation media. As a separation medium, PVAL homopolymer has shown poor resolutions probably due to its very strong hydrogen-bonding characteristics, resulting in extensive self-aggregation. On the other hand, poly(vinyl alcohol-co-vinyl acetate) and poly(vinyl alcohol-co-1-vinyl-2-pyrrolidone) (PVAL-VP), both with a degree of polymerization of approximately 3.0x10(3), have been found to give excellent electropherograms with good resolutions for the analysis of double-stranded (ds)DNA. PVAL-VP, with hydrolytic stability in high and low pH regions, has also yielded fair electropherograms for single-stranded (ss)DNA under neutral and alkaline conditions, although further investigation is essential in order to increase the resolutions necessary for DNA sequencing analysis. The separations obtained under alkaline conditions have shown significantly shorter retention times, one-third of that for the current commercial separation media, due to the higher ionization of phosphate groups in the DNA chains.

DNA↗
PubMed2003

Comparison of various implicit solvent models in molecular dynamics simulations of immunoglobulin G light chain dimer.

The present study tests performance of different solvation models applied to molecular dynamics simulation of a large, dimeric protein molecule. Analytical Continuum Electrostatics (ACE) with two different parameter sets, older V98 and new V01, and Effective Energy Function (EEF) are employed in molecular dynamics simulation of immunoglobulin G (IgG) light chain dimer and variable domain of IgG light chain. Results are compared with explicit solvent and distance dependent dielectric constant (DDE) calculations. The overall analysis shows that the EEF method yields results comparable to explicit solvent simulations; however, the stability of simulations is lower. On the other hand, the ACE_V98 model does not seem to achieve the accuracy or stability expected in nanosecond timescale MD simulation for the studied systems. The ACE_V01 model greatly improves stability of the calculation; nonetheless, changes in radius of gyration and solvent accessible surface of the studied systems may indicate that the parameter set still needs to be improved if the method is supposed to be used for simulations of large, polymeric proteins. Additionally, electrostatic contribution to the solvation free energy calculated in the ACE model is compared with a numerical treatment of the dielectric continuum model. Wall clock time of all simulations is compared. It shows that EEF calculation is six times faster than corresponding ACE and 50 times faster than explicit solvent simulations.

Computer Simulation↗
PubMed1997

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

Acetone↗
PubMed2004

Gibbs adsorption isotherm combined with Monte Carlo sampling to see action of cosolutes on protein folding.

Driven by conditions set by smaller solutes, proteins fold and unfold. Experimentally, these conditions are stated as intensive variables--pH and other chemical potentials--as though small solutes were infinite resources that come at an externally varied free energy cost. Computationally, the finite spaces of simulation allow only fixed numbers of these solutes. By combining the analytic Gibbs adsorption isotherm with the computational Monte Carlo sampling of polymer configurations, we have been able to overcome an inherent limitation of computer simulation. The idea is to compute analytically the free energy changes wrought by solutes on each particular configuration. Then numerical computation is needed only to sample the set of configurations as efficiently as when no bathing solute is present. For illustration, the procedure is applied to an idealized two-dimensional heteropolymer to yield lessons about the effect of cosolutes on protein stability.

Adsorption↗
PubMed1996

Low nanogram range quantitation of diglycerides and ceramide by high-performance liquid chromatography.

A method for ceramide (CER) and diradylglycerol (DG) determination after normal-phase HPLC separation was developed. The free oxydril group of ceramide and diradylglycerol is coupled to the carboxylic group of the fluorescent label (+)-6-methoxy-alpha-methyl-2 naphthaleneacetic acid (NAP), using as catalytic agents 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide. The use of NAP-free acid instead of the halide-activated form ensures higher stability of the reagent, lower reaction temperatures, and improved yield and reproducibility. The yield of the reaction is greater than 90% after a period of 3 h at the temperature of -20 degrees C. Over 85% of the starting material is recovered at the end of HPLC separation. The lower detection limit is below 5 ng for CER and 150 ng for DG. Under the conditions employed in the assay, no significant hydrolysis of triglycerides, sphingolipids, or phospholipids occurs and the esterification reaction is not affected by components of crude lipid extracts. Since separation and/or purification steps are not required, cellular levels of CER and DG can be easily and rapidly measured.

Cell Line↗
PubMed1992

Fluorescent rotors and their applications to the study of G-F transformation of actin.

New fluorescent rotor molecules having hydrophilic functional groups, which are derivatives of p-(N,N-dialkylamino)benzylidenemalononitrile, were synthesized. Their properties as fluorescent rotors were confirmed by an observation of solvent viscosity-dependent fluorescence. Incorporation of hydrophilic groups into the molecules increased the solubility of fluorescent rotors in aqueous media; the application of the compounds to biochemical systems became feasible as a consequence. To demonstrate this applicability, we attempted to monitor the G-F transformation of rabbit skeletal muscle actin with these newly synthesized compounds. All the compounds carrying a malononitrile moiety showed greater fluorescence in F-actin. Among them, 1-(2-hydroxyethyl)-6-[(2,2-dicyano)vinyl]-2,3,4-trihydroquinoli ne gave the best result by the criteria of the difference in fluorescence quantum yield for G- and F-actin, solubility, and stability of the compound. The method has the major advantage of not requiring covalent modification of actin.

Actins↗
PubMed1989

The interaction of 5,5-dimethyl-1-pyrroline-N-oxide with human myeloperoxidase and its potential impact on spin trapping of neutrophil-derived free radicals.

Activation of human neutrophils leads to secretion of myeloperoxidase (MPO) with resulting generation of several oxidant species including OCl-. Spin trapping techniques employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are being applied increasingly to the investigation of free radical production by in vitro and in vivo experimental systems which contain neutrophils. Because such knowledge is critical to the interpretation of these data, we examined the impact of MPO and MPO-derived oxidants on DMPO spin adduct formation and stability. Addition of increasing concentrations of OCl- to DMPO yielded a number of EPR-detectable products including DMPO-OH. However, the concentration of OCl- required was in excess of that expected under physiologic conditions. Addition of purified human MPO and H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO/H2O2 appeared to both generate and destroy DMPO spin adducts. Neutrophils stimulated with phorbol myristate acetate or opsonized zymosan generated large DMPO-OOH and DMPO-OH peaks as well as small DMPO-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 peak amplitudes but increased that of DMPO-OOH. These data suggest that MPO-derived oxidants likely have little impact on the nature of EPR spectra resulting from DMPO spin trapping of free radical species following neutrophil stimulation. Because MPO oxidants did appear to react with DMPO the ability of DMPO to protect a biologic target from in vitro MPO injury was examined. DMPO (greater than 10 mM) significantly decreased MPO/H2O2/Cl- -mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.

Cyclic N-Oxides↗
PubMed1993

Detection of alkaloids in foods with a multi-detector high-performance liquid chromatographic system.

A general screening method for alkaloid drugs in foods is described based on high-performance liquid chromatography with ultraviolet detection at three wavelengths, followed by fluorescence and electrochemical detectors in series. The chromatographic conditions include an ion-pairing reagent, which makes it possible to chromatograph acidic and basic drugs with one screen. Relative response ratios were determined from the peak areas of the alkaloids on the basis of all the detector signals. These ratios were used to create a "fingerprint" of the drugs and to predict the identity of an unknown component in a sample matrix. The fluorescence and electrochemical detectors allowed a detection limit for many of the alkaloids which would not be attainable with the ultraviolet detector alone. Typical detection limits with the electrochemical detector were 5-50 ng/ml and with the fluorescence detector 5-500 ng/ml, while the ultraviolet detector had detection limits of 1-20 micrograms/ml. The spiking concentrations in the relative response ratio experiments were approximately five times above the lowest detection limit. The extraction method investigated for orange juice yielded recoveries for most alkaloids of 80-100%. A stability study of ergot alkaloids in various food matrices demonstrated degradation, depending on the matrix, temperature, and duration of the experiment.

Alkaloids↗