Suitable Purity Research Articles

In Vitro Enzymatic Characterization of Near Full Length EGFR in Activated and Inhibited States

The epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein with an extracellular ligand-binding region and a cytoplasmic tyrosine kinase. Ligand binding activates the tyrosine kinase, which in turn initiates signaling cascades that influence cell proliferation and differentiation. EGFR activity is essential for normal development of many multicellular organisms, and inappropriate activation of EGFR is associated with multiple human cancers. Several drugs targeting EGFR activity are approved cancer therapies, and new EGFR-targeted therapies are being actively pursued. Much of what is known about EGFR structure and function is derived from studies of soluble receptor fragments. We report here an approach to producing an active, membrane-spanning form of EGFR of suitable purity, homogeneity, and quantity for structural and functional studies. We show that EGFR is capable of direct autophosphorylation of tyrosine 845, which is located on its kinase activation loop, and that the kinase activity of EGFR is approximately 500-fold higher in the presence of EGF vs the inhibitory anti-EGFR antibody cetuximab. The potencies of the small molecule EGFR kinase inhibitors erlotinib and lapatinib for various forms of EGFR were measured, and the therapeutic and mechanistic implications of these results considered.

Stoichiometry and Absolute Quantification of Proteins with Mass Spectrometry Using Fluorescent and Isotope-labeled Concatenated Peptide Standards

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.

Recovery of hydrocarbons from mixtures containing C 3H 6, C 3H 8 and N 2 using NaX membranes

The separation of light hydrocarbon (C 3H 6 and C 3H 8)/N 2 mixtures was investigated using faujasite membranes synthesized on α-Al 2O 3 disks. The motivation for studying this separation stems from the existence of vent streams in polyolefin production plants that contain these gases. These streams are usually flared and, as result, their valuable hydrocarbon content is lost. Permeation measurements of equimolar binary mixtures (C 3H 6/N 2 and C 3H 8/N 2) were carried out as a function of permeation temperature, feed mixture composition and total feed pressure. The maximum separation factors for C 3H 6/N 2 and C 3H 8/N 2 equimolar mixtures were ∼28.4 (with C 3H 6 flux 1.93 mmol m −2 s −1) and ∼8.1 (with C 3H 8 flux 1.34 mmol m −2 s −1), respectively and have been observed at 90 °C. As the total feed pressure was increased from 1 to 3.5 atm the permeation fluxes of all gases increased and the separation factor decreased. In particular, the separation factor of C 3H 6/N 2 dropped from ∼28 to ∼13 and of C 3H 8/N 2 from ∼8 to ∼5. Permeation experiments were carried out using a ternary mixture having a simulated composition of a typical polypropylene vent stream (12.5% C 3H 6/12.5% C 3H 8/75% N 2). The permeation results indicate that only C 3H 6 can be recovered from such a mixture. This result is very important because C 3H 6 is the valuable monomer and if recovered with suitable purity it can be recycled back to the polymerization reactor.

Isolation of DNA for RAPD analysis from dry leaf material of someHesperis L. specimens

An improved protocol for the isolation of DNA from dry material of someHesperis specimens is described. The isolated DNA is suitable for random amplification of polymorphic DNA (RAPD) analysis. Different DNA extraction protocols were examined to determine which might yield DNA from dry leaf tissue ofHesperis specimens. The methods examined include the protocols with hexadecyltrimethylammonium bromide (CTAB) described by Doyle and Doyle (1987); sodium dodecyl sulfate (SDS) by Dellaporta et al. (1983); and CTAB and SDS, the modified minipreparation, by Dellaporta et al (1983). None of these procedures yielded DNA of suitable purity for RAPD assay. We established an improved procedure involving CTAB and enzymatic digestion of proteins and RNA. The recovery of DNA with an average yield of 25 mg/g of leaf material was possible with this procedure. RAPD bands, which could be used to distinguish amongHesperis specimens, were generated.

Parallel synthesis of tri- and tetrasubstituted ureas from carbamoyl imidazolium salts.

A method for producing tri- and tetrasubstituted ureas from carbamoyl imidazolium salts is presented. Carbamoyl imidazolium salts are prepared from the reaction of N,N carbonyldiimidazole (CDI) with secondary amines, followed by alkylation with iodomethane. These stable salts can be stored for extended periods and are effective electrophilic carbamoylation reagents. Primary and secondary amines add to carbamoyl imidazolium salts at room temperature to give tri- and tetrasubstituted ureas in excellent yields. This reaction was used to synthesize ureas using both liquid-liquid extraction and solid-phase extraction (cation exchange) purification techniques. Liquid-liquid extraction affords the product ureas more cleanly than cationic exchange. A series of urea compounds were synthesized using parallel synthesis techniques in high yields and with suitable purity for routine in vitro biological tests. These studies validate the utility of carbamoyl imidazolium salts as useful building blocks for combinatorial library synthesis.

Microwave-Assisted Parallel Synthesis of a 2-Aryl-1H-Isoindole-1,3-Dione Library

An efficient parallel synthesis of a representative 28-member library of phthalimides is described. Parallel chromatography afforded the library members in suitable purity and with high yields.

Characterization of phospho-olivines as materials for Li-ion cell cathodes

Solid State Reaction was employed to prepare phospho-olivines LiMPO4 (where M=Fe, Co) as pure phase and LiNiPO4 in presence of foreign phases, as cathodic materials for lithiumions batteries. The relationship between structural, morphological and electrochemical properties were investigated in the case of LiFePO4. Structural investigation has been carried out by means of X-ray powder diffraction (XRPD) and Rietveld refinement. The influence on the morphology of annealing temperature, different flowing gas mixture and addition of ascorbic acid during the synthesis, has been analysed via scanning electron microscopy. The electrochemical cycling performances on LiFePO4 showed to be positively affected by the modifications of the experimental conditions. Cyclic voltammetry showed a good reversibility during insertion-extraction mechanism, in particular in presence of additives. LiCoPO4 and LiNiPO4 are interesting as high voltage cathode materials for Li-ion batteries and have been taken into account, but their electrochemical operating conditions are still to be optimised. In the case of LiNiPO4 it is very difficult to obtain, by solid state synthesis, suitable purity powders, having a grain size small enough to exploit it usefully as cathodic material for Li-ion cells.

Clinical allergy to cocamidopropyl betaine: reactivity to cocamidopropylamine and lack of reactivity to 3-dimethylaminopropylamine.

7 patients allergic to cocamidopropyl betaine (CAPB) were detected by positive patch test reactions to Tegobetaine L7TM. These patients were then asked to participate in further testing to its potential impurities, cocamidopropylamine and 3-dimethylaminopropylamine (DMAPA). 4 of the 7 patients were tested to purified CAPB and cocamidopropylamine, with 2 reacting to the purified betaine on allergy patch testing and 3 reacting to cocamidopropylamine 0.1%. At another date, 6 of the 7 were successfully recalled for testing to DMAPA in the presence of sodium lauryl sulfate (SLS) or pure CAPB; 1/6 reacted to DMAPA in water, but only at very high concentrations, at least 3 orders of magnitude higher than that to which skin exposure would occur from use of products containing CAPB. Both SLS and CAPB increased the number of reactions recorded to high levels of DMAPA. However, positive reactions to much lower concentrations of DMAPA (>or=100 ppm) were found in only 1/6 subjects and then only in the presence of the irritant SLS. 0/6 reacted to pure CAPB alone. Taken together, these results suggest that DMAPA is unlikely to be an important contact allergen in CAPB of appropriate quality. They also confirm that CAPB of suitable purity, where levels of both cocamidopropylamine and DMAPA are minimized, is unlikely to trigger reactions in those ostensibly allergic to the material.

Synthesis of Zirconia-Based Solid Electrolyte Powders by the Coprecipitation Technique

The coprecipitation technique has been used in order to obtain zirconia-magnesia and zirconia-ceria powders with suitable purity and sinterability, respectively. To accomplish those requests, some processing variables have been systematic studied and optimized. Similar solid solutions have been prepared by the powder mixing technique for comparison purposes. Inductively coupled plasma, thermal neutron activation, nitrogen adsorption/desorption, thermal analyses and scanning electron microscopy techniques have been used for powder characterization. Cylindrical pellets were prepared by uniaxial and cold isostatic pressing. The characterization of the sintered pellets have been carried out by impedance spectroscopy, scanning electron microscopy and X-ray diffraction techniques. Zirconia-magnesia ceramics with a high degree of purity have been obtained, in agreement with impedance spectroscopy results that show no grain boundary blocking effects. For the zirconia-ceria system, sintered pellets with 98% of the theoretical density, and 0.5 μm average grain size have been obtained without using any comminution or special drying processes.

Isolation and characterization of highly purified rat intestinal intraepithelial lymphocytes

The study of intestinal intraepithelial lymphocytes (IEL) has been hindered by the difficulty of isolating a population of lymphocytes which is free of epithelial cell or lamina propria cell contaminants and representative of the in vivo population of IEL in both phenotype and function. We describe an improved technique for the extraction and purification of IEL from the proximal small intestine of the rat. This technique rapidly and reproducibly isolates 5–10×10 6 IEL/rat with 90–95% purity and viability without the use of enzymes which affect lymphocyte function. The resulting cell population, which is 75% αβ T cell receptor (TCR) +, 70% CD8 +, and 33% CD4 + T cells, and only 5% B cells and 2% macrophages, is of suitable purity to allow for flow cytometric analysis of the entire population of cells without requiring gating on lymphocytes. IEL are comprised of a unique T cell repertoire in that 27% of cells co-express the CD4 and CD8 molecules, but only 11% of CD4 + cells co-express CD45RC. All CD4 + cells express the αβTCR, but 9% of IEL are CD8 +CD4 −αβTCR −. The adhesion molecules α 4 integrin and l-selectin are expressed on 57% and less than 1% of IEL, respectively. The isolated IEL population contains mRNA for IL-1α, IL-1β, IL-1R, IL-1RA, IL-2, IL-6R, IFN-γ, TGF-α, TGF- β 1, and TNF-α. Mesenteric lymph node cells (MLNC) were examined in parallel. This technique allows for the isolation of rat IEL appropriate for phenotypic analysis by flow cytometry and for cytokine analysis by reverse transcription/polymerase chain reaction.

Supercritical fluid extraction of benzodiazepines in solid dosage forms

Several benzodiazepines were separated from the matrices of their standard dosage forms using supercritical fluid extraction. Extracts were further processed by gas chromatography-mass spectrometry and/or Fourier transform infrared spectrometry. Extracts of suitable purity and quantity for mass spectral analysis were easily obtainable from even the lowest concentration dosage forms of the drugs tested.

A plasmid-based approach for the synthesis of a histidine biosynthetic intermediate

A novel plasmid DNA construct has been developed for use in the synthesis of a histidine biosynthesis intermediate,N 1 -[(5''-phospho-β-D-ribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (5'-ProFAR, 1). The approach allows for a convenient, millimole-scale preparation of the bis-ribosyl nucleotide and provides material of suitable purity for spectral analyses and biochemical studies. A highly enriched source of the first three enzymes in the histidine bisynthetic pathway has been developed by constructing a recombinant DNA plasmid which directs the overexpression of these proteins

Genome typing of mouse adenoviruses.

Restriction endonuclease cleavage site analysis was used to differentiate between mouse adenovirus (MAV) types 1 and 2 strains. Viral DNA of suitable purity and quantity for multiple enzymatic digestions was obtained from cloned CMT-93 mouse tumor cells infected with each type of MAV. Clear differences between the MAV-1 (FL) and MAV-2 (K87) genomes were observed after cleavage with restriction enzymes such as BglII, EcoRI, and PaeR7. Fast electrophoresis of DNA fragments in miniature agarose slab gels allowed rapid and unequivocal identification of the MAV strains. This relatively simple and accurate method should be quite useful to determine the different modes of transmission of mouse adenoviruses and their presence in various animal populations.

A study of the purification of metallurgical grade silicon

Purification of metallurgical grade silicon to solar grade silicon has been investigated by acid leaching of pulverised silicon and by copper alloying processes. The acid leaching process involved refluxing silicon with aqua regia and HF. Leaching with HF was found to be more efficient than leaching with aqua regia. The copper alloying process, which involved melting silicon in the presence of copper, electroextraction of copper (for reuse) from CuSi alloys and acid washing of anode slime, was also found to yield silicon of suitable purity.

Secretory granules.

Although considerable knowledge has been acquired concerning the chemical nature of insulin and its pathway of biosynthesis, relatively little is known about the other proteins which constitute the insulin secretory granule. This, in part, has been a consequence of the difficulties of working with pancreatic islets as a tissue source and the attendant problems of obtaining sufficient material of suitable purity for experimental purposes. The scope for further investigation is perhaps best illustrated by the fact that of the 150 or so insulin granule proteins which may be visualized by electrophoresis the identity of less than 10 has been established. It is clear that some of the proteins will be shared by other granule types in accordance with the common cellular pathways involved in granule morphogenesis and exocytotic secretion. Equally though, it is also apparent that many of the peptides of the insulin granule are tailored for specific functions within the B-cell.

Tritiation of 1,2,3‐benzenetriol (pyrogallol)

AbstractThe synthesis of 4,6‐2H2‐1,2,3‐benzenetriol from 1,2,3‐benzenetriol was possible under both acid and base catalyzed conditions. However, under simulated physiological conditions it was observed that the label underwent exchange and was lost. Using a three step synthesis, 5‐2H‐1,2,3‐benzenetriol was synthesized and the label was found to be stable to exchange under the same conditions. Using this synthesis, 5‐3H‐1,2, 3‐benzenetriol was synthesized with a specific activity of 7.6 mCi/mmole and of suitable purity for use in metabolic studies.

Identification of canine urinary metabolites of the antiprotozoal agent 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole

Metabolite fractions from the urine of a dog dosed with 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436) were obtained by the use of high-performance liquid chromatography. These fractions were of suitable purity for structural elucidation. Data obtained by mass spectrometry and NMR spectroscopy allowed the identification of seven major metabolites of this drug. Biotransformation in each case involved hydroxylation (mono or di) of the hexahydrobenzisoxazole ring.

A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis

A simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.

A comparison of the structure of hamster pancreatic insulin and the insulin extracted from a transplantable hamster islet-cell carcinoma

Insulin has been isolated from pancreases of the Syrian hamster and from a transplantable islet-cell tumor of the hamster. Acid/ethanol extraction, ether precipitation, ion exchange and gel filtration chromatography gave preparations of suitable purity for structural studies. Using trypsin cleavage, automatic Edman degradation and manual Edman degradation, a complete sequence of the pancreatic insulin B chain was determined. By automatic Edman degradation, the amino-terminal 10 residues of the pancreatic A chain were assigned and the sequence of carboxy-terminal eleven residues could be deduced by homology to other mammalian and avian insulins. The sequence assigned to hamster insulin A chain is identical to that of the rat, mouse and spiny mouse. The sequence of hamster insulin B chain is identical to rabbit and spiny mouse B chain. In terms of protein evolution, hamster insulin thus appears to occupy an intermediate position between rabbit and rat insulins. Amino acid composition, tryptic peptide composition and partial sequence analysis of the hamster tumor insulin showed no differences from hamster pancreatic insulin.

Chapter 17 Purity and Stability of Radiochemical Tracers in Autoradiography

Publisher Summary High-resolution autoradiographic techniques are of special value in cell biology for locating the site of action of substances in cells. Autoradiography makes it possible to trace substances labeled with a radioisotope in animal or plant organisms macroscopically and down to subcellular structures. The methods employed are normally of three kinds—namely, macroscopic, microscopic, and electron microscopic autoradiography. This chapter highlights some of the basic problems that occur and that can influence interpretation of the facts derived from the use of autoradiography in biological investigations and in cell biology, in particular. In addition to the normal considerations of the appropriate photographic emulsion and the sample preparation techniques to be used for the autoradiographs, various other points that need to be carefully considered in the design of a tracer investigation to achieve useful valid data include correct choice of radionuclide, appropriate molar specific activity and chemical form of the tracer, and checking of the tracer compound for suitable purity. To obtain meaningful results from radiochemical tracer experiments using autoradiography to make the results visible, it is necessary to ensure that the radiochemical is of suitable radiochemical purity and free of impurities likely to give false results. For some studies, it may be necessary to establish the pattern of labeling in the tracer compound to interpret the autoradiographic data.