The tritium labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate--polyacrylamide slab gels.

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [³H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.     

High-performance liquid chromatographic analysis of dianhydrogalactitol in plasma by derivatization with sodium diethyldithiocarbamate

A high-performance liquid chromatographic method is described for measuring submicrogram quantities of dianhydrogalactitol, a promising anti-neoplastic agent, in plasma. The drug is derivatized directly in plasma with sodium diethyldithiocarbamate to form a bis(dithiocarbamoyl) ester which absorbs UV light at 254 nm (α_m 17,000). The derivatized product is then extracted quantitatively into chloroform and separated by normal phase chromatography (μBondpak CN column). Dianhydrogalactitol concentration below 50 ng/ml of plasma can be detected in the eluent.     

Gel electrophoresis of fluorescent labeled cyanogen bromide cleavage products at the submicrogram level.

A simple method is described for visualizing submicrogram amounts of the cyanogen bromide cleavage products of proteins on sodium dodecyl sulfate-polyacrylamide gels. The peptide fragment mixture is conjugated with the fluorogenic reagent 2-methoxy-2,4,-diphenyl-3-(2H)-furanone prior to electrophoresis. The fluorescent peptide bands are visible under ultraviolet light, thus avoiding the need for fixation and staining. The determination of the structural homology of two immunologically related proteins is presented to illustrate this methodology.     

Development of a dispersive liquid-liquid microextraction method for spectrophotometric determination of barbituric acid in pharmaceutical formulation and biological samples

In this paper, a novel and simple method for the determination of trace amounts of barbituric acid in water and biological samples was developed by using dispersive liquid–liquid microextraction (DLLME) techniques combined with spectrophotometric analysis. The procedure is based on color reaction of barbituric acid with p-dimethylaminobenzaldehyde and extraction of the color product using the DLLME technique. Some important parameters such as reaction conditions and the type and volume of extraction and dispersive solvents as well as the extraction time were investigated and optimized in detail. Under the optimum conditions, the calibration graphs were linear over the range of 5.0 to 200 ng ml⁻¹ with limit of detection of 2.0 ng ml⁻¹. Relative standard deviation for five replicate determinations of barbituric acid at 50 ng ml⁻¹ concentration level was calculated to be 1.64%. Average recoveries for spiked samples were determined to be between 94% and 105%. The proposed method was applied for the determination of barbituric acid in pharmaceutical formulation and biological samples.     

A silver stain for the detection of nanogram amounts of tRNA following two-dimensional electrophoresis

Three ultrasensitive protein silver-staining methods have been compared with respect to the detection of tRNA in polyacrylamide gels. The method of Sammons (D. W. Sammons, L.D. Adams, and E.E. Nishizawa (1981) Electrophoresis 2, 135-141) has been shown to have remarkable sensitivity, with a detection limit of 0.3 ng tRNA/mm2, allowing the two-dimensional fractionation of submicrogram amounts of bulk tRNA. The application of this technique to developmental and differentiation problems and other areas where the amounts of nonradioactive tRNA available are limited is anticipated.     

Zonal rate centrifugation of proteoglycans in sucrose gradients

A simple reverse-phase chromatographic system for separating deoxyribonucleoside monophosphates is described. Using isocratic elution at room temperature, clear separation of seven of the deoxyribonucleoside monophosphates that occur in either procaryotic or eucaryotic DNAs can be achieved in less than 2 h. Thus, this method allows a sensitive and rapid analysis of submicrogram quantities of ³²P-labeled deoxyribonucleoside monophosphates derived from DNA labeled in vivo with ³²P_i or from DNA labeled enzymatically in vitro at the 5′ or 3′ ends. The suitability of the method for studying methylation of mammalian DNAs is illustrated by presenting examples of its application to (a) quantitation of major and minor nucleotides in newly synthesized DNA, (b) determination of the specificity of in vitro methylation of DNA, and (c) quantitation of the extent to which specific restriction endonuclease sites are methylated in vivo.     

A Quantitative Microanalysis of Bacterial Endotoxin Using [3H]-Labeled L-Glycero-D-mannoheptitol as a Marker

Quantitative microanalysis of bacterial endotoxin was performed using [³H]-labeled L -glycero-D -mannoheptitol (LD -Heptitol) as a marker. Several different amounts of authentic L -glycero-D -mannoheptose (LD -Heptose) were reduced with 20 μg of cold NaBH₄ containing 2 μg of NaB³H₄ (40 Ci/mmol) in 20 μ1 of 1 mM NaOH at 4 C for 48 hr. The product, [1-3H]-labeled LD -Heptitol, has high specific activity, and was purified by HPLC and detected using a liquid-scintillation counter. As little as 50 pg of LD -Heptose was detectable, and the radioactivity increased dose-dependently in the 100 pg to 80 ng range tested. More than 2 ng of Salmonella abortus equi endotoxin could be accurately determined by this method. It is possible to detect 50 pg of endotoxin by this method, if 100% hot material (NaB³H₄) is used for [³H]-labeling.     

ZUR ENERGIEBEDÜRFTIGEN Ca2+-AKKUMULATION IN MITOCHONDRIEN AUS RATTENHIRN

—(1) The properties of a preparation of functional intact rat brain mitochondria are described and the resulting fraction is enzymatically characterized. (2) These mitochondria are able to accumulate Ca²⁺ in a respiration-dependent reaction without additions of P_i and/or adenine nucleotides. (3) As criteria the increased acidity of the incubation medium accompanying the energy-dependent Ca²⁺ accumulation, its inhibition by rotenon and Antimycin A, the acceleration of respiration and the redox change of the pyridine nucleotides were recorded and Ca²⁺ accumulation by the mitochondria was determined by complexemetric methods. (4) The maximum Ca²⁺ accumulation by rat brain mitochondria amounts to only 25 per cent of that by rat liver mitochondria under similar conditions (on the base per mg protein); after addition of 1 mm -ADP (without P_i) the comparable value was about 50 per cent.     

Radioimmune assay of tubulin applied to oligomers, synaptic membranes, and plants

A radioimmune assay for microtubule protein, tubulin, is described, in which unknown amounts of native or denatured tubulin can be quantitated by the ability to compete with pure [¹²⁵I]tubulin for rabbit antibodies produced against purified bovine brain tubulin. The assay is used to demonstrate that crude extracts of mouse brain contain negligible amounts of 30–36S tubulin oligomers under conditions where purified tubulin forms substantial amounts of such structures. Also, the particulate fraction of osmotically shocked and sonicated brain synaptosomes contains negligible tubulin antigenic activity. By contrast, soluble extracts of soybean, especially rapidly dividing regions of the plant, were found to contain significant amounts of cross-reacting material, providing further evidence for the conservative evolutionary nature of this ubiquitous and important protein.     

Bacterial synthesis,purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels

An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives (¹⁵N-, ¹⁵N-/¹³C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly α-helical conformation. The existence of cross-peaks for all glycines of the ¹⁵N-HSQC NMR spectra as well as relatively small line widths (∼20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.     

Nitrate sorption in the profile of an acid soil

Sorption of NO _inf3 ^sup– by different horizons of a highly weathered, acid tropical soil was measured in laboratory batch experiments. Sorption was found to increase with depth, ranging from small amounts in the 0–15 cm layer to amounts that would be roughly equivalent to 25 to 50% of the NO _inf3 ^sup– in the 90–120 cm layer at water and NO _inf3 ^sup– contents commonly found under field conditions. Calculations, based on sorption isotherms, demonstrated how sorption may be important for managing N in a tropical acid soil. Sorption of Cl^– was also found in the range of 0.1 and 2.0 mol m^–3. In this range of concentrations sorption of NO _inf3 ^sup– and chloride were found to be independent, suggesting that anion exchange sites were far from saturated.Contribution from the Department of Soil, Crop and Atmospheric Sciences, New York State College of Agriculture and Life Sciences, Cornell University, Ithaca, NY 14853. SCAS paper No. 1726. This research is part of the TropSoils program.Contribution from the Department of Soil, Crop and Atmospheric Sciences, New York State College of Agriculture and Life Sciences, Cornell University, Ithaca, NY 14853. SCAS paper No. 1726. This research is part of the TropSoils program.     

Thiocyanate inhibition of protein iodination by the chloramine-T method and a rapid method for measurement of low levels of thiocyanate

Thiocyanate interferes with iodination of protein by the chloramine-T method. A 50% reduction of ¹²⁵I incorporation into protein results from SCN⁻ at 10⁻⁵ . A simple, rapid, and convenient method of detection of low levels of SCN⁻ in protein solutions by its colored complex with Fe³⁺ is described.     

Quantitation of sorafenib and its active metabolite sorafenib N-oxide in human plasma by liquid chromatography–tandem mass spectrometry

A simple and rapid method with high performance liquid chromatography/tandem mass spectrometry is described for the quantitation of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma. A protein precipitation extraction procedure was applied to 50 μL of plasma. Chromatographic separation of the two analytes, and the internal standard [²H₃¹³C]-sorafenib, was achieved on a C₁₈ analytical column and isocratic flow at 0.3 mL/min for 4 min. Mean within-run and between-run precision for all analytes were <6.9% and accuracy was <5.3%. Calibration curves were linear over the concentration range of 50–10,000 ng/mL for sorafenib and 10–2500 ng/mL for sorafenib N-oxide. This method allows a specific, sensitive, and reliable determination of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma in a single analytical run.     

Progesterone synthesis,secretion and metabolism by human teratoma-derived cell-lines

The synthesis, secretion and metabolism of progesterone have been examined in six human teratoma-derived cell lines with the objective of determining if they exhibit trophoblast-related or other specific steroidogenic functions. Progesterone was synthesised in nanogram amounts (per 10⁶ cells/day) by the cell line SuSa, as measured by radioimmunoassay, and in lesser amounts by line LICR-LON HX-39. Lines Tera 1, Tera 2, T₃B₁ and PA-1 did not secrete detectable progesterone. All teratomas, however, metabolized added progesterone in microgram amounts (per 10⁶ cells/day). In all cases the major metabolite was a polar compound, identified by reversed phase HPLC, TLC and GC-MS as 3β, 6α-dihydroxy-5α-pregnan-20-one. This pattern of metabolism was not confined to the teratomas as equivalent amounts of this polar metabolite were formed by cultures of adult differentiated human epithelial and fibroblast cells. When progesterone and its metabolites, separated by HPLC, were included in the estimation, the Δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity of SuSa was equivalent to 47ng pregnenolone (3β-hydroxy-5-pregnen-20-one) metabolised/mg protein/day, that of HX-39 to 9ng/mg protein/day and those of other teratomas to <3.5ng/mg protein/day.     

Thermo-osmoregulation of Heat-Labile Enterotoxin Expression by <Emphasis Type="Italic">Escherichia coli</Emphasis>

Abstract:Enterotoxigenic Escherichia coli causes diarrhea by producing several virulence factors including heat-labile enterotoxin (LT). LT is maximally expressed at 37°C. The histone-like nucleoid structuring protein (H-NS) appears to inhibit LT expression by binding to a downstream regulatory element (DRE) at low temperatures. An hns⁺ E. coli strain, X7026, carrying an LT–beta-galactosidase translational fusion plasmid (pLT-lac) was shown to be responsive to varying amounts of sodium chloride (NaCl) as well as sucrose or lithium chloride. Maximal responsiveness to the various osmolytes was obtained with cells grown at 37°C under microaerophilic conditions. Temperature-osmotic upshift experiments demonstrate LT expression is thermo-osmoregulated. pLT-lac was tested in an hns strain or its congenic hns⁺ strain for its response to NaCl. LT expression is elevated in the hns strain regardless of NaCl concentration and retains its osmoresponsiveness. The response of the DRE deletion plasmid (pLT-lacNC) to NaCl is similar to that of the undeleted plasmid.     

Stoichiometry for guanine nucleotide binding to tubulin under polymerizing and nonpolymerizing conditions

The maximal stoichiometry for [³H]GTP binding to depolymerized tubulin with saturating amounts of added [³H]GTP is 0.4 mol/110,000 g protein. In contrast, 1 mol of radioactive nucleotide is incorporated into microtubules as a result of polymerization with [³H]GTP. The different stoichiometries result from a difference in the nucleotide binding properties of ring protein under polymerizing and nonpolymerizing conditions: ring protein at 0 °C is devoid of binding activity but binds added radioactive guanine nucleotide during microtubule assembly. The radioactive nucleotide which is incorporated into rings during microtubule assembly is not displaced by excess GDP, although it is at a site which is distinct from the N site.     

AN ISOTOPIC MICROMETHOD FOR THE MEASUREMENT OF CHOLINESTERASE ACTIVITY IN INDIVIDUAL CELLS

—A microisotopic method for measuring acetylcholinesterase activity in isolated cells is described. The assay employs [¹⁴C]acetylcholine and can measure 7 × 10^-12 moles of acetylcholine hydrolysed/hr in 50-150 samples per experiment. The method described has been applied to the measurement of cholinesterase activity in individual sympathetic ganglion cells of the cat. It has been shown that under standard conditions the substrate has complete access to the enzymatic site.     

Assay for l-pipecolate oxidase activity in human liver: detection of enzyme deficiency in hyperpipecolic acidaemia

A direct assay method is described for l-pipecolate oxidase. The assay uses NaHSO₃ to trap the $\text{L}\text{-α-amino[}{}^3\text{H]adipate}$δ-semialdehyde (AAS) formed as a direct reaction product of l-pipecolate oxidase from l-[³H]pipecolic acid. The adduct so formed was separated from the substrate on Dowex 50 (H⁺) column. The product was identified as [³H]AAS by amino acid analysis after breaking down the adduct by boiling under acidic conditions. The assay is simpler and more specific than fluorometric methods; it is also more sensitive; requiring at most 16 μg of liver peroxisome-enriched protein per assay. We have used this assay procedure to detect l-pipecolate oxidase in skin fibroblasts obtained from a control subject and from patients of hyperpipecolic acidaemia and Zellweger syndrome and found that this enzyme activity is present in the control, but absent or decreased in the patients with the peroxisomal disorders.     

Inhibition of Cellular Protein Synthesis by Simultaneous Pretreatment of Host Cells with Fowl Plague Virus and Actinomycin D: a Method for Studying Early Protein Synthesis of Several RNA Viruses

A method is described for analysis of viral protein synthesis early after infection when minute amounts of viral proteins are effectively concealed by large amounts of produced host-specific proteins. The method is superior to a radioimmune assay, since all virus-induced proteins can be measured independent of their immunological reactivity. Host-specific protein synthesis can be suppressed by infection with fowl plague virus. Addition of actinomycin D 1.25 h postinfection does not prevent this suppression, but it does block effectively the formation of fowl plague virus-specific proteins. Such cells synthesize only small amounts of cellular proteins, as revealed by polyacrylamide electrophoresis. They can be superinfected with several different enveloped viruses, however, without significant diminution of virus yields. In pretreated cells the eclipse is shortened for Semliki Forest virus, Sindbis virus, and vesicular stomatitis virus, but prolonged for Newcastle disease virus. The onset of protein synthesis, specific for the superinfecting virus, could be clearly demonstrated within 1 h after superinfection. At this time, in cells superinfected with Semliki Forest virus, great amounts of NSP 78 (nonstructural protein; molecular weight, 78 × 10³) and reduced amounts of the core protein C could be demonstrated. The precursor glycoprotein NSP 68 is followed by a new polypeptide, NSP 65; three proteins with molecular weights exceeding 100 × 10³ were observed which are missing later in the infectious cycle. Similar results were obtained after superinfection with Sindbis virus. The formation of a new polypeptide with a molecular weight of about 80 × 10³ was detected. After superinfection with vesicular stomatitis virus or Newcastle disease virus the formation of new proteins, characteristic for the early stage of infection, was not observed.     

Transformation of Phaffia rhodozyma by electroporation

Summary The transformation of Phaffia rhodozyma by electroporation was better than the one by the lithium chloride (LiCl) or spheroplast transformation methods. The influence of several parameters on transformation efficiency was studied. Electroporation conditions of 1200 Volts, 50 Farads and 2000 Ohms were proved successful. A maximum of 1.5 × 10³ transformants per 100 ng of DNA was reached.