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The measurement of androgen receptors in human prostatic tissue utilizing sucrose density centrifugation and a protamine precipitation assay.

Methods have been established for the measurement of androgen receptors in the cytosol and crude nuclear extract of human prostatic tissue using sucrose density gradient centrifugation and a protamine precipitation assay. These studies have demonstrated the presence of a high affinity, heat labile protein that binds preferentially to dihydrotestosterone and that is present only in androgen target tissues. In studies of prostatic tissue from 15 patients sucrose density gradient centrifugation appeared more sensitive than the protamine precipitation assay in the identification of androgen receptors. These findings appear promising and suggest that in the near future a reliable assay may be available for the measurement of androgen receptors in prostatic carcinoma.

Centrifugation, Density Gradient↗

Ammonium sulfate precipitation as a tool for the study of androgen receptor proteins in rat prostate and mouse kidney.

Ammonium sulfate precipitation has been used for the separation of bound and free steroids in rat prostate and mouse kidney cytosol equilibrated with tritiated androgens. A high affinity, low capacity binding protein has been identified in the 35% saturation precipitate. Biochemical and physiological data indicate that this protein is identical with the previously described 8-10 S androgen receptor. It has been demonstrated that this receptor protein binds 17 beta - hydroxy-5alpha-androstan-3-one (DHT) and testosterone in both tissues. The apparent dissociation constant (Kd) of the prostatic receptor for DHT and of the renal receptor for testosterone is 1-2 nM. The number of binding sites equals 57 and 23 fmoles/mg protein in prostate and kidney respectively. Dterminations of apparent inhibition constants (Ki) for 26 steroidal and non-steroidal compounds suggest that the binding sites in these tissues is similar or identical.

Ammonium Sulfate↗

Influence of impurity ions and magnetic field on the properties of freshly precipitated calcium carbonate.

Static magnetic field (MF) effects on the properties of freshly precipitated calcium carbonate have been investigated in the presence of impurity ion Mg(2+), Fe(2+), or SO4(2-). One or both solutions, CaCl2 and Na2CO3, were exposure to MF (0.5T) for 20min at 20 degrees C. Then calcium carbonate was precipitated and zeta potential, pH and light absorbance (lambda=543.3 nm) were measured. The same parameters were also determined for the reference systems in which the solutions were not MF-treated. It was found that in all the systems tested MF effects as determined by the above mentioned parameters had appeared. They depended on the kind of the impurity ion present, as well as on which solution, CaCl2, Na2CO3 or both, MF interacted. For example, if Mg(2+) ion was present in CaCl2 solution, the largest shift in the zeta potential toward higher positive values was observed if Na2CO3 was MF-treated (e.g. from 2 to 12mV) and the same was true as for the maximum in the light absorbance and the pH increase. Interestingly, if (CaCl2 + Mg(2+)) was MF-treated pH of the slurry had decreased. Moreover, a correlation between above mentioned MF effects and the entropy of hydration of the ions has also been found. This points to the changes in the hydrating water structure caused by magnetic field.

Calcium Carbonate↗

The mechanism of metals precipitation by biologically generated alkalinity in biofilm reactors.

Microbial strains, characterized by increased tolerance and ability to grow in metal bearing wastewaters as well as by effective metal sequestering capability by both active (bioaccumulative) and passive (biosorptive) processes, were tested as inoculum for metal laden wastewater treatment systems. Their capacity to grow in metal bearing wastewater, using an easily available and inexpensive carbon source such as acetate, was studied in batch experiments. Two principal conclusions were drawn: (1). Growth was observed for all the strains examined suggesting that the strains can be acclimated to metals bearing wastewaters. (2). Solution pH increased from neutral to alkaline values during growth (pH(initial)=7, pH(final)=10). The later was observed systematically for all strains. Metal precipitation, due to the metabolically generated alkalinity is expected as a result.Supporting evidence for this hypothesis was provided during the operation of two pilot moving bed sand filters treating two different metal bearing wastewaters. Acetate was used as carbon source to support the growth and maintenance of microbial biomass on the sand grains of the filters. The characteristics of the sludge produced from the operation of the pilot plants were subsequently studied in the laboratory. Both sludges were significantly loaded with large amounts of metals.A mechanism of metal precipitation induced by the metabolically generated alkalinity, when acetate is used as carbon source, could be proposed as the main process responsible for the metals sequestering inside the moving bed sandfilter reactor.

Adsorption↗

Precipitation of carbonates by Nesterenkonia halobia in liquid media.

We investigated the precipitation of carbonates by Nesterenkonia halobia in a liquid medium at different concentrations of salts and incubation times. N. halobia only produced crystals at salt concentrations of 2.5%, 7.5% and 15%. At 20% salt concentration no crystal formation was observed. Calcite, aragonite and dolomite were precipitated in different quantities, depending on the salinity of the medium and incubation time. Scanning and transmission electron microscopy, microanalysis and electron diffraction were all used to study in detail the morphology, composition and internal structure of the bioliths. We propose a mechanism for biolith formation involving both biological and inorganic processes.

Carbonates↗

Protein precipitation with polyethylene glycol.

Polyethylene glycol is a nondenaturing water-soluble polymer whose ability to precipitate protein from aqueous solution can be qualitatively understood in terms of an excluded volume mechanism. The increment in PEG concentration required to effect a given reduction in solubility is unique for a given protein-polymer pair, being insensitive to solution conditions and primarily dependent on the size of the protein and polymer. Selective manipulation of the solubility of specific proteins through control of their state of association or ligand environment can potentially remove some of the empiricism otherwise involved in fractional precipitation. Adequate methods for removing the polymer are available.

Chemical Precipitation↗

Kinetics of hydroxyapatite precipitation at pH 10 to 11.

The mechanism and kinetics of hydroxyapatite (HAP) precipitation from aqueous solution at the conditions of pH 10-11 and at calcium ion concentrations of more than 0.5 mol/l were investigated. The results show that the reaction undergoes the following process: transferring from octacalcium phosphate (OCP) to amorphous calcium phosphate (ACP) rapidly, and then from ACP to calcium-deficient hydroxyapatite (DAP) and HAP. DAP was the non-stoichiometric material that ACP converts to HAP. Reaction temperature greatly affects the reaction rate of the conversion from ACP to HAP. It takes about 24 h to form pure-phase HAP at 25 degrees C while it takes only 5 min at 60 degrees C. The temperature also has a great deal of influence on the particle size and morphology of precipitated hydroxyapatite. The reaction of transformation from ACP to DAP was second order and the activation energy was 95 kJ/mol (22.7 kCal/mol) and therefore it was inferred that the conversion reaction was a surface control process.

Calcium Phosphates↗

Fabrication of poly(phosphoester) nerve guides by immersion precipitation and the control of porosity.

Immersion precipitation was employed as a method for the fabrication of polymeric conduits from P(BHET-EOP/TC), a poly(phosphoester) with an ethylene terephthalate backbone, to be applied as guidance channels for nerve regeneration. Coatings of various porosities could be obtained by immersing mandrels coated with a solution of the polymer in chloroform into non-solvent immersion baths, followed by freeze or vacuum-drying. The porosity of the coatings decreased with an increase in polymer molecular weight, drying time before precipitation and concentration of polymer solution. The effects of these parameters can be rationalized by employing ternary phase diagrams, where porosity is directly related to the degree of phase separation available to the system before gelation occurs. To afford improved porosity control, a new system was developed which employed the contrasting phase-separation behavior of P(BHET-EOP/TC)/chloroform solution in methanol and water. As water is essentially a non-solvent for the polymer, the demixing boundary of the P(BHET-EOP/TC)-CHCl3-H2O system is located close to the polymer-solvent edge of the phase diagram, while that of the P(BHET-EOP/TC)-CHCl3-MeOH system is located further away. A mixture of methanol and water allows the demixing boundary to be shifted to intermediate coordinates. By immersing P(BHET-EOP/TC) coatings in immersion baths containing different ratios of water and methanol, then gradually titrating the bath with methanol to a concentration of 70% (v/v) methanol, surface porosities ranging from 2 to 58% could be achieved.

Biocompatible Materials↗

La ions in precipitated hydroxyapatites.

Hydroxyapatites were synthesized by precipitation from an aqueous solution with La3+ (0-0.75%) and with carbonate (0-6.1%) at controlled pH 7.0. Uptake of La3+ was 90-95% complete. Relatively low Ca/P (1.54-1.63) ratios were attributed to nonstoichiometry. Carbonate in samples was identified by IR spectroscopy as B-type carbonate. Lattice parameters of the hexagonal apatite structure were not affected by the La3+ content. Noncarbonated samples heated to 800 degrees C transform partially to beta-Ca3(PO4)2. Thermogravimetric analysis showed release of 0.4 mol adsorbed and 1 mol crystalline water up to 400 degrees C and decomposition of carbonate up to 900 degrees C in the samples. Luminescence data obtained for Gd-containing hydroxyapatites prove that Gd3+ ions are not incorporated in the precipitated hydroxyapatite. These findings suggest that, in the La-containing samples, La3+ is surface absorbed and not incorporated in hydroxyapatite.

Calcium↗

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.

Cerebrospinal Fluid Proteins↗

Intrahepatocellular erythrocyte inclusions and increased calcium precipitation in canine endotoxic shock.

AIMS: To investigate the electron microscopic localization of membrane-bound and exchangeable calcium with specific calcium precipitation techniques during endotoxic shock in the dog. METHODS: Ten pentobarbital anesthetized, mechanically ventilated, and paralyzed dogs were studied. Six dogs received 2 mg/kg E. coli endotoxin i.v. followed by a continuous 0.9% saline infusion to restore and maintain baseline cardiac filling pressures. Four dogs served as time-matched controls. Each experiment lasted for 3 h. After the completion of study, the livers of four endotoxic and two control dogs were fixed by perfusion of 3% glutaraldehyde via the portal vein. Liver sections were then prepared for electron microscopy and calcium localization studies. RESULTS: Hepatocytes of endotoxic animals completely lost their plasma membrane-bound calcium. The most severely damaged cells showed extensive "blebbing" of the plasma membrane and contained numerous cytoplasmic erythrocyte inclusions. Endotoxin administration also caused excessive calcium precipitation inside hepatocytes in areas with pronounced sinusoidal damage. CONCLUSIONS: In this acute model of fluid-resuscitated endotoxic shock in dogs, the use of specific calcium localization techniques enables the demonstration of disturbances in hepatocellular calcium handling, which appear to be closely related to structural alterations of the hepatocyte cell membrane. Erythrocyte uptake by hepatocytes is a previously undescribed phenomenon in canine endotoxic shock and may serve as an additional histologic marker of ultrastructural cell (membrane) damage.

Animals↗

Protein precipitation: a common etiology in neurodegenerative disorders?

Molecular genetic analyses have elucidated a class of inherited neurodegenerative disorders caused by expanded CAG repeats encoding polyglutamines (e.g. Huntington disease and Machado-Joseph disease). Proteins containing expanded polyglutamine repeats appear to precipitate by self-aggregation and, as a result, produce a core disease-related phenotype: neuronal cell death or degeneration. In other neurodegenerative disorders, such as Alzheimer disease, prion disease, Parkinson disease and amyotrophic lateral sclerosis, precipitation of abnormal proteins is also now considered to play a key role. These observations might lead to the elucidation of universal mechanisms for neurodegeneration and to effective treatments for many neurodegenerative disorders.

Chemical Precipitation↗

A reaction-transport model for calcite precipitation and evaluation of infiltration fluxes in unsaturated fractured rock.

The percolation flux in the unsaturated zone (UZ) is an important parameter addressed in site characterization and flow and transport modeling of the potential nuclear-waste repository at Yucca Mountain, NV, USA. The US Geological Survey (USGS) has documented hydrogenic calcite abundances in fractures and lithophysal cavities at Yucca Mountain to provide constraints on percolation fluxes in the UZ. The purpose of this study was to investigate the relationship between percolation flux and measured calcite abundances using reactive transport modeling. Our model considers the following essential factors affecting calcite precipitation: (1) infiltration, (2) the ambient geothermal gradient, (3) gaseous CO(2) diffusive transport and partitioning in liquid and gas phases, (4) fracture-matrix interaction for water flow and chemical constituents, and (5) water-rock interaction. Over a bounding range of 2-20 mm/year infiltration rate, the simulated calcite distributions capture the trend in calcite abundances measured in a deep borehole (WT-24) by the USGS. The calcite is found predominantly in fractures in the welded tuffs, which is also captured by the model simulations. Simulations showed that from about 2 to 6 mm/year, the amount of calcite precipitated in the welded Topopah Spring tuff is sensitive to the infiltration rate. This dependence decreases at higher infiltration rates owing to a modification of the geothermal gradient from the increased percolation flux. The model also confirms the conceptual model for higher percolation fluxes in the fractures compared to the matrix in the welded units, and the significant contribution of Ca from water-rock interaction. This study indicates that reactive transport modeling of calcite deposition can yield important constraints on the unsaturated zone infiltration-percolation flux and provide useful insight into processes such as fracture-matrix interaction as well as conditions and parameters controlling calcite deposition.

Calcium Carbonate↗

Detection of precipitating and complement consuming antibodies by enzyme linked immune sorbent assay in pigeon breeders' disease.

The sera of patients with pigeon breeders' disease usually contain both precipitating and complement-fixing antibodies to antigens in pigeon droppings or pigeon serum. These antibodies are not equivalent. Both types of antibody could be detected by means of an ELISA technique, using either pigeon serum or pigeon dropping antigens in the solid phase. The good correlation between the ELISA results and the complement consumption assay indicates that the ELISA technique may represent a one-step assay for assessing both precipitating and complement-consuming IgG-class antibodies, thereby identifying both symptomatic patients and breeders potentially at risk. IgM antibodies appeared to be of minor significance in the serology of pigeon breeders' disease.

Alveolitis, Extrinsic Allergic↗

A pyridine-thiol ligand with multiple bonding sites for heavy metal precipitation.

There are immediate concerns with current commercial ligands that are used for heavy metal precipitation, especially the limited arrays of bonding sites. Previous research has indicated that not only do commercial reagents lack sufficient bonding criteria, but they also fail to provide long-term stability as ligand-metal complexes. For this reason, we have developed a pyridine-based thiol ligand (DTPY) which not only offers multiple bonding sites for heavy metals but also should form stable metal-ligand precipitates. In this study, we used the divalent metals cadmium and copper to model the reactivity and pH stability of divalent metal complexes with the DTPY ligand. Using inductively-coupled plasma spectrometry (ICP), results indicate that a 50.00ppm (parts per million) copper solution, pH of 4.5, can be reduced to below the ICP detection limits of 0.00093ppm (>99.99% removal), and a 50.00ppm cadmium solution, pH of 6.0, can be reduced to 0.06ppm (99.88%).

Chemical Precipitation↗

Using the magnitude of PSA bounce after MRI-guided prostate brachytherapy to distinguish recurrence, benign precipitating factors, and idiopathic bounce.

PURPOSE: To identify events that precipitated a prostate-specific antigen (PSA) bounce and characterize the magnitude, duration, and time to PSA bounce after MRI-guided prostate brachytherapy. METHODS AND MATERIALS: Between 1997 and 2001, 186 patients with low-risk prostate cancer underwent MRI-guided permanent 125I source implantation, with or without external beam radiotherapy. A PSA bounce was defined as a >or=15% elevation in PSA compared with the most recent value, followed by a decline to a level at or less than the prebounce value. At the time of PSA measurement, data were prospectively collected on whether the patient had recent ejaculation, ongoing radiation proctitis, or recent instrumentation. RESULTS: A total of 115 patients (61.8%) had a total of 156 PSA bounces. Of these, 36 patients had PSA bounces associated with ejaculation, proctitis, or instrumentation, and 79 experienced idiopathic PSA bounces (not associated with a precipitating event). The magnitude of the PSA bounce was significantly lower for the idiopathic PSA bounce (0.6 ng/mL) compared with that associated with ejaculation (p = 0.003), proctitis (p <0.0001), or instrumentation (p = 0.007). Patients with biopsy-proven local recurrence had a median PSA elevation of 1.2 ng/mL, significantly higher (p = 0.006) than the magnitude of the idiopathic PSA bounce, but not significantly different from the magnitude of the PSA bounce due to ejaculation, proctitis, or instrumentation. CONCLUSION: In patients treated with MRI-guided prostate brachytherapy, recent ejaculation, instrumentation, or ongoing radiation proctitis can cause a transient increase in PSA, the magnitude of which is significantly higher than that for idiopathic PSA bounce, but is similar to that in patients with recurrent disease.

Aged↗

Quantitative determination of thalidomide in human serum with high-performance liquid chromatography using protein precipitation with trichloroacetic acid and ultraviolet detection.

A validated and precise reversed-phase high-performance liquid chromatographic method for the determination of thalidomide in serum, with phenacetin as an internal standard, is described. Protein precipitation, using trichloroacetic acid, was used for clean-up. The aliquot was chromatographed on a octadecyl column, using an eluent composed of 250 ml 0.01 M potassium dihydrogenphosphate, adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 750 ml methanol. Ultraviolet detection was used at an operation wavelength of 220 nm. Hydrolytic degradation was prevented during analysis by acidification of samples with the precipitation reagent. Thalidomide and phenacetin were found to have retention times of 7.9 and 15.0 min, respectively. Recoveries ranging from 79 to 84% were found for both components, with reproducibility relative standard deviations of 0.8-3% and repeatability coefficients of 1.2-3%. A mean correlation coefficient of 0.9995 was found for the linear calibration curve (n=2) of thalidomide with limits of quantitation of 0.222-21 mg/l. The method appeared to be feasible for pharmacokinetic studies with thalidomide.

Blood Proteins↗

Automated protein precipitation by filtration in the 96-well format.

The use of automated protein precipitation by filtration in the 96-well format as a rapid sample preparation technique for high throughput bioanalysis using liquid chromatography tandem mass spectrometry is reported. A robotic sample processor is used to aspirate sequentially a plasma sample and acetonitrile separated by air gaps. These are then mixed by being dispensed into individual channels of a 96-well filter block. The resulting supernatant is separated from the precipitated plasma proteins by the application of gentle vacuum using a custom manifold. The filtered supernatants are collected into a deep well microtitre plate, evaporated to dryness using a heated 96-well dry down station and reconstituted in water prior to analysis. The efficiency of the extraction procedure is measured by the Lowry method for determining protein concentration. This method was used to optimise both the volume and the order of reagent addition, and to compare several prototype 96-well filter blocks. Using the optimised procedure a specific, precise and accurate method was developed for the beta-agonist salbutamol in rabbit plasma with a calibration range of 1 to 100 ng/ml from 100 microl of sample.

Albuterol↗