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PubMed1984

Preparation, analysis and application of [99mTc]Human Albumin Microspheres ([99mTc]HAM) for lung scanning.

Kits were developed for the sterile labelling of Human Albumin Microspheres with 99mTc. The microspheres were prepared, sieved to get the desired particle size and autoclaved for sterilization. The spheres were treated with stannous chloride and the pH of the suspension was adjusted to 3.7 with phosphate buffer. After freeze-drying the contents of single reaction vials from different batches were reacted with 99mTc and the radiochemical yield (higher than 97%) was determined. The HAM kits were stable and the stability of [99mTc]HAM was followed for 5 h. Lung and liver uptake in mice was determined to be about 90 and 1% respectively. The preparation of [99mTc]HAM is performed in a single step process and excellent human lung scans were obtained.

Adult↗
PubMed1994

Significant contribution of arginine-112 and its positive charge of Pseudomonas putida cytochrome P-450cam in the electron transport from putidaredoxin.

Cytochrome P-450cam of Pseudomonas putida is a prototype of various eukaryotic cytochrome P-450 molecules. Arg-112 located on the surface of this protein is highly conserved among various other cytochromes P-450. In this study, we constructed mutant genes for P-450cam in which Arg-112 was replaced by Gln or Glu, expressed them in Escherichia coli and purified the mutant proteins. Their enzymic activities were analyzed in the reconstituted system to determine the function of Arg-112. Kd values for d-camphor of Arg112-Gln and Arg112-Glu were much the same as those of the wild-type enzyme, whereas Kd values for the oxidized form of putidaredoxin, which is an acidic protein and is the redox partner of P-450cam, were 240 and 530 microM, respectively. These values are 8 and 19 times larger than that of the wild-type enzyme (28 microM), thereby indicating lower affinities of the mutant enzymes for the oxidized putidaredoxin. Reaction rate constants for reduction by the reduced form of putidaredoxin, measured using the stopped flow method, were 45.5, 9.0 x 10(-3) and 9.0 x 10(-4) s-1 for the wild type, Arg112-Gln and Arg112-Glu, respectively. Thus, Arg-112 of P-450cam plays an important role in the interaction with putidaredoxin and in the high efficiency of the electron transfer; the positive charge of the residue seeming to contribute to the process. The yields in Escherichia coli, the heme contents in the purified fractions and heat stability of the mutant proteins were lower than those of the wild type enzyme, suggesting that Arg-112 of P-450cam is also important for stability of P-450cam.

Arginine↗
PubMed1983

Isolation and characteristics of bovine pituitary secretory granules.

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measureable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both growth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10(-5) M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.

Animals↗
PubMed2006

A disposable on-chip phosphate sensor with planar cobalt microelectrodes on polymer substrate.

Disposable microsensors on polymer substrates consisting of fully integrated on-chip planar cobalt (Co) microelectrodes, Ag/AgCl reference electrodes, and microfluidic channels have been designed, fabricated, and characterized for phosphate concentration measurement in aqueous solution. The planar Co microelectrode shows phosphate-selective potential response over the range from 10(-5) to 10(-2)M in acidic medium (pH 5.0) for both inorganic (KH(2)PO(4)) and organic (adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphates (ADP)) phosphate compounds. This microfabricated sensor also demonstrates significant reproducibility with a small repeated sensing deviation (i.e. relative standard deviation (R.S.D.)<1%) on a single chip and a small chip-to-chip deviation (i.e. R.S.D.<2.5%). Specifically, while keeping the high selectivity, sensitivity, and stability of a conventional bulk Co-wire electrode, the proposed phosphate sensor yields advantages such as ease of use, cost effectiveness, reduced analyte consumption, and ease of integrating into disposable polymer lab-on-a-chip devices. The capability to sense both inorganic and organic phosphate compounds makes this sensor applicable in diverse areas such as environmental monitoring, soil extract analysis, and clinical diagnostics.

Biosensing Techniques↗
PubMed2005

Determination of sixteen polycyclic aromatic hydrocarbons in aqueous and solid samples from an Italian wastewater treatment plant.

A robust procedure for the determination of 16 US EPA PAHs in both aqueous (e.g. wastewaters, industrial discharges, treated effluents) and solid samples (e.g. suspended solids and sludge) from a wastewater treatment plant (WWTP) is presented. Recovery experiments using different percentages of organic modifier, sorbents and eluting solvent mixtures were carried out in Milli-Q water (1000 mL) spiked with a mixture of the PAH analytes (100 ng/L of each analyte). The solid phase extraction (SPE) procedures applied to spiked waste water samples (1000 mL; 100 ng/L spiking level) permitted simultaneous recovery of all the 16PAHs with yields >70% (6-13% RSD). SPE clean up procedures applied to sewage and stabilized sludge extracts, showed percent recoveries in the range 73-92% (7-13% RSD) and 71-89% (7-12% RSD), respectively. The methods were used for the determination of PAHs in aqueous and solid samples from the WWTP of Fusina (Venice, Italy). Mean concentrations, as the sum of the 16PAHs in aqueous and suspended solid samples, were found to be approx. in the 1.12-4.62 microg/L range. Sewage and stabilized sludge samples contained mean PAH concentrations, as sum of 16 compounds, in the concentration range of 1.44-1.26 mg/kg, respectively. Extraction and clean up procedures for sludge samples were validated using EPA certified reference material IRM-104 (CRM No. 912). Instrumental analyses were performed by coupling HPLC with UV-diode array detection (UV-DAD) and fluorescence detection (FLD).

Chromatography, High Pressure Liquid↗
PubMed2005

Development of an enteric-coated, layered multi-particulate formulation for ileal delivery of viable recombinant Lactococcus lactis.

Layering of recombinant hIL-10 producing Lactococcus lactis (L. lactis Thy12) on inert carriers is a promising technique for the preparation of a multi-particulate formulation of viable, hIL-10 producing L. lactis. To improve viability after layering and storage, L. lactis Thy12 was layered in different matrices (10% skim milk and/or 2.5, 5, 10% inulin). After layering, the highest viability was obtained in the 10% skim milk supplemented with 5% inulin matrix (8.7%). However, upon storage, 10% skim milk alone yielded the highest viability. Thereby, layered L. lactis Thy12 showed superior long term stability in comparison with freeze-dried L. lactis Thy12. The layering process was performed during 3h without encountering technical problems, with good layer consistence and constant viability. Enteric properties were obtained with a 30% Eudragit L30D-55 or 15% Eudragit FS30D coating and maintained during an initial six months storage period (-20 degrees C/20% RH). After in vitro simulation of the gastric stage, only 5% of the bacteria remained viable in Eudragit L30D-55 coated pellets, contrary to 85% in Eudragit FS30D coated pellets, indicating its superior protective capacity against gastric fluid. After eight months storage (-20 degrees C), 80% of the initial L. lactis Thy12 remained viable in the Eudragit FS30D coated pellets.

Chemistry, Pharmaceutical↗
PubMed2006

Preparation, characterization and surface study of poly-epsilon caprolactone magnetic microparticles.

Magnetic microparticles (MMP) have shown to be applied in increasing applications in various fields of biotechnology and medicine. One of their most promising utilization is the magnetic resonance imaging (MRI) in which superparamagnetic substances as magnetite are used in a nanometric size (less than 30 nm) and encapsulated within locally injected biodegradable microparticles. In this paper, magnetite has been encapsulated in polymer-based microparticles. The MMP have been prepared by an emulsion evaporation method. The different parameters influencing the particles size were investigated. The size was found to decrease as the stirring speed or the stabilizer amount (to certain limit) increases. The encapsulation efficacy was more than 90% yielding a magnetite loading of up to 30%, w/w. The X-ray photoelectron spectroscopy (XPS) showed less than 2% of iron atoms at the microparticles surface. The zeta potential response of MMP towards pH variation was very similar to that of magnetite-free microparticles confirming the encapsulation of magnetite within the microparticles. X-ray diffraction assays showed that magnetite crystalline structure was conserved after emulsification and MMP formation. Vibration simple magnetometer (VSM) showed a superparamagnetic profile of the MMP with a magnetic saturation increasing with the increased magnetite amount in the microparticles. These magnetic microparticles can enable clinicians to control microparticles distribution after a local administration in tumors by MRI. They can also be administered to target a defined tumor area by focusing a magnetic field on the surfaces covering the cancerous tissue.

Colorimetry↗
PubMed2006

Spectroscopic and electronic structure studies of the role of active site interactions in the decarboxylation reaction of alpha-keto acid-dependent dioxygenases.

The alpha-ketoglutate (alpha-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require Fe(II), alpha-KG and dioxygen for catalysis, with the alpha-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the alpha-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an Fe(IV)O intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O(2) to generate this species is the decarboxylation of the alpha-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the alpha-keto acid to Fe(II) and the presence of a 5C Fe(II) site for the O(2) reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate alpha-KG coordination and a 5C Fe(II) site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an approximately 10(5)-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n-->pi( *) transition of the HPPD/Fe(II)/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the alpha-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO(2) is disfavored.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2005

Sensitive determination of nucleic acids using organic nanoparticle fluorescence probes.

This paper describes the preparation of organic nanoparticles by reprecipitation method under sonication and vigorous stirring. Transmission electron microscopy (TEM) was used to characterize the size and size distribution of the luminescent nanoparticles. Their average diameter was about 25 nm with a size variation of +/-18%. The fluorescence decay lifetime of the nanoparticles also was determined on a self-equipped fluorospectrometer with laser light source. The lifetime (approximately 0.09 micros) of nanoparticles is about three times long as that of the monomer. The nanoparticles were in abundant of hydrophilic groups, which increased their miscibility in aqueous solution. These organic nanoparticles have high photochemical stability, excellent resistance to chemical degradation and photodegradation, and a good fluorescence quantum yield (25%). The fluorescence can be efficiently quenched by nucleic acids. Based on the fluorescence quenching of nanoparticles, a fluorescence quenching method was developed for determination of microamounts of nucleic acids by using the nanoparticles as a new fluorescent probe. Under optimal conditions, maximum fluorescence quenching is produced, with maximum excitation and emission wavelengths of 345 and 402 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.4-19.0 microgml(-1) for calf thymus DNA (ct-DNA) and 0.3-19.0 microgml(-1) for fish sperm DNA (fs-DNA). The corresponding detection limits are 0.25 microgml(-1) for ct-DNA and 0.17 microgml(-1) for fs-DNA. The relative standard deviation of six replicate measurements is 1.3-2.1%. The method is simple, rapid and sensitive with wide linear range. The recovery and relative standard deviation are very satisfactory.

Animals↗
PubMed2002

Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl beta-D-maltoside.

Here we report a fast, simple purification for thermophilic F1F0 ATP synthase (TF1F0) that utilizes a cocktail of stabilizing reagents and the detergent n-dodecyl beta-D-maltoside to yield enzyme with an ATPase activity of 41 micromol/min/mg, 2.5-fold higher than that previously reported. ATPase activity was 80% inhibited by the F0-reactive reagent dicyclohexylcarbodiimide, indicating that F1-F0 interactions were largely intact. To measure ATP-driven proton pumping activity, purified TF1F0 was incorporated into liposomes, and the ATP-induced change in internal pH was measured using the fluorescent probe pyranine. In the presence of valinomycin, a maximum ATP-driven deltapH of 0.8 units was obtained. To measure ATP synthesis activity, TF1F0 was incorporated into liposomes with the light-dependent proton pump bacteriorhodopsin. Proteoliposomes were illuminated to generate an electrochemical gradient, after which ADP and inorganic phosphate were added to initiate ATP synthesis. A steady state ATP synthesis activity of 490 nmol/min/mg was achieved after an initial approximately 30-min lag phase.

Adenosine Triphosphate↗
PubMed2003

Design and selection of ligands for affinity chromatography.

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential.

Chromatography, Affinity↗
PubMed1992

Purification of the vesamicol receptor.

The vesamicol receptor (VR) present in cholinergic synaptic vesicles isolated from the electric organ of Torpedo was solubilized in cholate detergent and stabilized with glycerol and a phospholipid mixture. The receptor was purified in 7% yield by hydroxylapatite, wheat germ lectin affinity, DEAE anion-exchange, and size exclusion chromatographies based on a [3H]vesamicol binding assay. A final specific binding of 4400 pmol/mg of protein was obtained. The cholate-solubilized VR complex exhibited variable aggregation states with particle molecular masses of 210-3500 kDa in different experiments. The purified VR exhibited very heterogeneous electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with very diffuse protein staining at about 240 kDa. No "classical" polypeptide or glycopeptide band was detected. One form of the SV1 epitope, which is characteristic of cholinergic synaptic vesicle proteoglycan, copurified precisely with the VR. The SV2 epitope, which is found in most neuronal and endocrine secretory vesicles, also closely purified with the VR. Substantially purified VR retained both enantioselectivity for (-)-vesamicol and a linked AcCh-binding site. This confirms the allosteric model for the VR in the AcCh transporter. The physicochemical properties of the VR and copurification of it with the SV1 epitope strongly suggest that the VR is associated with cholinergic vesicle proteoglycan. A second proteoglycan that is not associated with the VR but which carries the SV1 and SV2 epitopes also was observed.

Acetylcholine↗
PubMed2005

pH- and metal ion-linked stability of the hemopexin-heme complex.

Thermal denaturation of the human hemopexin-heme complex was investigated under a variety of solution conditions to identify factors that influence heme release. The midpoint temperature for the transition between the folded and folded states, T(m), of the hemopexin-ferriheme complex exhibits a significant dependence on pH. When the pH is reduced from 7 to 5 (50 mM BisTris buffer and 50 mM NaCl), T(m) decreases by approximately 23 degrees C despite the relatively higher chloride concentration that tends to stabilize the protein. The thermal stability of the hemopexin-ferroheme complex was examined at pH 7.4 to yield a T(m) that is 3.2 degrees C lower than that of the hemopexin-ferriheme complex under identical conditions. The effect of transition metal ions, which hemopexin has recently been shown to bind [Mauk, M. R., Rosell, F. I., Lelj-Garolla, B., Moore, G. R., and Mauk, A. G. (2005) Biochemistry 44, XXXX-XXXX], was also considered. Cu(2+) and Zn(2+) had the greatest effect, reducing T(m) for the transition by 4.8 and 6.5 degrees C, respectively, relative to the value for the protein in the absence of metal ions [T(m) = 64.9 degrees C [10 mM sodium phosphate buffer (pH 7.4)]]. These metal ions also interfered significantly with the recovery of the native state from the unfolded protein when the protein on returning to 20 degrees C. The current results demonstrate how the conditions within the endosomes of hepatocytes (pH approximately 5.0, [Cl(-)] approximately 60 mM) and the potential presence of transition metal ions or heme iron reduction contribute to the membrane receptor-mediated process of heme release from hemopexin.

Buffers↗
PubMedDate not supplied

Spectrophotometric determination and computational evaluation of the rates of hydrolysis of 9-amino-substituted acridines.

Aminoacridines have a long history in the drug and dye industries and display a wide range of biological and physical properties. Despite the historical relevance of 9-aminoacridines, there have been few studies investigating their stability. 9-Aminoacridines are known to hydrolyze at the C9-N15 bond, yielding acridones. In this study, the pH-dependent hydrolysis rates of a series of 9-substituted aminoacridines are investigated. In addition, ground-state physical properties of the compounds are determined using ab initio quantum mechanics calculations to gain insight into the forces that drive hydrolysis. An analysis of the bond orders, bond dissociation energies, and conformational energies show that the rate of hydrolysis depends on two main factors: delocalization across the C9-N15 bond and steric effects. The computational results are applied to explain the change in experimental rates of hydrolysis going from primary to secondary and to tertiary substituted 9-aminoacridines. In the case of tertiary substituted amines, the calculations indicate the C9-N15 bond is forced into a more gauche-like conformation, greatly diminishing delocalization (as shown by reductions in bond orders and bond energy), which leads to rapid hydrolysis. A model of intramolecular hydrogen bonding is also presented, which explains the increased rate of hydrolysis observed for highly substituted compounds under acidic conditions.

Acridines↗
PubMed2005

Contributions of organic peroxides to secondary aerosol formed from reactions of monoterpenes with O3.

The role of organic peroxides in secondary organic aerosol (SOA) formation from reactions of monoterpenes with O3 was investigated in a series of environmental chamber experiments. Reactions were performed with endocyclic (alpha-pinene and delta3-carene) and exocyclic (beta-pinene and sabinene) alkenes in dry and humid air and in the presence of the OH radical scavengers: cyclohexane, 1-propanol, and formaldehyde. A thermal desorption particle beam mass spectrometer was used to probe the identity and volatility of SOA components, and an iodometric-spectrophotometric method was used to quantify organic peroxides. Thermal desorption profiles and mass spectra showed that the most volatile SOA components had vapor pressures similar to pinic acid and that much of the SOA consisted of less volatile species that were probably oligomeric compounds. Peroxide analyses indicated that the SOA was predominantly organic peroxides, providing evidence that the oligomers were mostly peroxyhemiacetals formed by heterogeneous reactions of hydroperoxides and aldehydes. For example, it was estimated that organic peroxides contributed approximately 47 and approximately 85% of the SOA mass formed in the alpha- and beta-pinene reactions, respectively. Reactions performed with different OH radical scavengers indicated that most of the hydroperoxides were formed through the hydroperoxide channel rather than by reactions of stabilized Criegee intermediates. The effect of the OH radical scavenger on the SOA yield was also investigated, and the results were consistent with results of recent experiments and model simulations that support a mechanism based on changes in the [HO2]/[RO2] ratios. These are the first measurements of organic peroxides in monoterpene SOA, and the results have important implications for understanding the mechanisms of SOA formation and the potential effects of atmospheric aerosol particles on the environment and human health.

Aerosols↗
PubMed2007

Convenient method to access new 4,4-dialkoxy- and 4,4-diaryloxy-diaza-s-indacene dyes: Synthesis and spectroscopic evaluation.

A straightforward method for the synthesis of original 4,4-dialkoxy- or 4,4-diaryloxy-diaza-s-indacenes (BODIPY) derivatives obtained by treatment of BODIPY 1 with various alcohols in the presence of AlCl3 is described. The novel compounds are characterized by spectroscopic properties similar to those of the parent BODIPY 1, absorption and emission spectra with similar band shapes, high molar absorption coefficients (epsilon lambda max approximately 80,000 M(-1) cm(-1)), and for most of them high fluorescence quantum yields (Phi exp from 0.52 to 0.71). Among all of the new compounds synthesized, the dye 2 h exhibits higher fluorescence quantum yield (0.71) and lifetime (4.09 ns) than compound 1 and a good chemical stability toward conditions compatible with biological cell-based assays.

Aza Compounds↗
PubMed2005

Adsorption of organic matter at mineral/water interfaces. 6. Effect of inner-sphere versus outer-sphere adsorption on colloidal stability.

The effects of the adsorption modes of several low molecular weight (LMW) organic anions (maleate, oxalate, and citrate) on the colloidal stability of corundum-water suspensions have been examined using electrokinetic and shear yield stress (tau(y)) measurements over a broad range of pH conditions and LMW organic anion concentrations. Consistent with previous studies, increasing concentrations of maleate, oxalate, and citrate progressively shift the electrokinetic isoelectric point and pH of the maximum shear yield stress (tau(y,max)) to more acidic conditions. Due to its predominant electrostatic driving force for adsorption, outer-spherically adsorbed maleate possesses a very limited ability to charge reverse the corundum-water interface or bind to the negatively charged corundum surface. By contrast, inner-spherically adsorbed oxalate and citrate can significantly charge reverse the corundum-water interface, with the extent of charge reversal being related to the relative binding strengths of the oxalate and citrate anions. Adsorbed maleate, oxalate, and citrate generate steric barriers to interparticle approach, leading to substantial reductions in the magnitude of tau(y,max) at low to intermediate concentrations of those LMW anions. At the highest anion concentrations investigated, however, increases in tau(y,max) are observed, and can be attributed to the formation of bridging Al(III)-organic surface precipitates, as suggested by in situ attenuated total reflectance Fourier transform infrared spectroscopic measurements of corundum-oxalate suspensions at high oxalate concentrations. The extent of precipitate formation is greatest for the corundum-oxalate system due to the strong dissolution-enhancing properties of the inner-spherically adsorbed oxalate anion (i.e., its ability to generate enhanced concentrations of dissolved Al(III) which can then participate in precipitate formation). The effects of the LMW organic anion adsorption modes on both the forms of the measured tau(y) versus pH data, and the ability to quantitatively compare tau(y) and zeta potential data measured at different corundum concentrations, are also discussed.

Journal Article↗
PubMed2001

Total synthesis of the microtubule-stabilizing agent (-)-laulimalide.

[structure: see text] The total synthesis of the potent microtubule-stabilizing anticancer agent (-)-laulimalide has been achieved in 27 steps and 2.9% overall yield. Notable features are the use of Jacobsen HDA chemistry for the enantioselective construction of the side chain dihydropyran, a diastereoselective aldol coupling using chiral boron enolate methodology, a Mitsunobu macrolactonization, and a Sharpless AE to introduce the epoxide onto des-epoxy-laulimalide.

Antineoplastic Agents↗