Method for improving the detection of viruses in fecal samples

A method is described that improved the detection of viruses in fecal samples by electron microscopy. The virus particles were concentrated, and much of the background debris was removed by adsorption of viruses on meat protein added to the fecal sample at a low pH and a low salt concentration. Viruses were eluted by raising the pH and the salt concentration. Further concentration was achieved by acid precipitation and vacuum dialysis.     

Method for improving the detection of viruses in fecal samples.

A method is described that improved the detection of viruses in fecal samples by electron microscopy. The virus particles were concentrated, and much of the background debris was removed by adsorption of viruses on meat protein added to the fecal sample at a low pH and a low salt concentration. Viruses were eluted by raising the pH and the salt concentration. Further concentration was achieved by acid precipitation and vacuum dialysis.     

Two methods for electrophoretic elution of proteins from polyacrylamide gels.

An apparatus is described which improves an earlier technique for eluting proteins from polyacrylamide-gel slabs by electrophoresis against a sucrose gradient. Another elution method where the components are concentrated electrophoretically in a collodion bag by altering the current density is described. This method enables the elution of small amounts of sample, free from disturbing background material arising from the gel, and also permits subsequent dialysis and ultrafiltration without transfer losses. It can be used with alkaline and acidic buffers and has been applied in the purification of human pituitary thyrotropin (TSH).     

Dynamic Dialysis: An Efficient Technique for Large-Volume Sample Desalting

Dialysis is a well-known technique for laboratory separation. However, its efficiency is commonly restricted by the dialyzer volume and its passive diffusion manner. In addition, the sample is likely to be precipitated and inactive during a long dialysis process. To overcome these drawbacks, a dynamic dialysis method was described and evaluated. The dynamic dialysis was performed by two peristaltic pumps working in reverse directions, in order to drive countercurrent parallel flow of sample and buffer, respectively. The efficiency and capacity of this dynamic dialysis method was evaluated by recording and statistically comparing the variation of conductance from retentate under different conditions. The dynamic method was proven to be effective in dialyzing a large-volume sample, and its efficiency changes proportionally to the flow rate of sample. To sum up, circulating the sample and the buffer creates the highest possible concentration gradient to significantly improve dialysis capacity and shorten dialysis time.     

Preparative separation of proteins using centrifugal precipitation chromatography based on solubility in ammonium sulfate solution

A preparative modification of the centrifugal precipitation chromatography (CPC) is described. The sample-loading capacity is improved in the present system by the use of convoluted tubing containing dialysis tubing instead of a dialysis membrane placed between a pair of disks equipped with mirror-imaged spiral grooves as in the original design. The system uses, basically, the same principle of as the original CPC, in that a concentration gradient of precipitant is generated under a centrifugal force field. The protein sample injected into the CPC column is exposed to an increasing concentration of the precipitant where it precipitates at various portions of the column according to its solubility. The gradient is then gradually lowered so that the sample undergoes dissolution and precipitation many times within the column; the proteins finally elute from the column according to their solubilities. A basic study was performed using this machine to separate human albumin and 3-globulin using ammonium sulfate (AS) as precipitant. Preliminary results indicate that this method can separate 500 mg of protein.     

A modified equilibrium dialysis method for studying fatty acid binding to proteins

A modified equilibrium dialysis method is described which is suitable for investigating the binding of fatty acids in the form of aqueous micellar dispersions to proteins. The method uses a permeant chromophore which complexes reversibly with free fatty acid within the dialysis bag. The concentration outside the dialysis bag is determined spectrophotometrically. Binding of oleic acid to bovine serum albumin is given as an example. A simplified analysis of fatty acid binding is given and used to indicate the potential of the method.     

Automated analysis of free and total concentrations of three antiepileptic drugs in plasma with on-line dialysis and high-performance liquid chromatography

A fully automated method for determination of the free and total concentration of drugs with a varying degree of protein binding is described. The antiepileptic drugs phenytoin, carbamazepine and phenobarbitone were chosen to demonstrate the utility of this technique. The method was based on the ASTED system and combined on-line equilibrium dialysis at 37°C with concentration of the dialysate on a trace enrichment column and HPLC determination with UV detection. The dialysis cell was a modification of the ASTED dialysis cell and 22% of the free concentration of the drugs were recovered in the recipient channel of the dialyser after 10 min of dialysis at 37°C. The free concentration, the total concentration as well as the drugs protein binding could be determined. The method was shown to be well suited for routine monitoring of the free and the total concentrations of the drugs in plasma from epileptic patients.     

A critical reappraisal of Waddell's technique for ultraviolet spectrophotometric protein estimation

Waddell's method of estimating protein concentration by the difference between spectrophotometric absorptions (215-225 nm) has been reexamined. Over limited ranges of total protein, a linear relation was found for ten purified proteins; the narrowest range was between 5 and 25 micrograms/ml. Using published extinction coefficients at 280 nm for these ten proteins, protein concentration at 280 nm correlated closely with the 215 nm/225 nm difference measurements (mean difference of 2.6%). Waddell's method also accurately determined the total protein in a mixture of proteins with widely varying individual 280-nm extinction coefficients. Biuret estimates of total protein in plasma or serum gave poor correlation with measurements by Waddell's method. Within protein concentration limits, Waddell's method was linear, narrow, and more variable, both for individual proteins and for protein mixtures, than previously reported. Within these limits, the method is probably as accurate a measure of total protein as measurement by nitrogen analysis, with the advantage of being nondestructive.     

Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B

A method has been described for the measurement of apoB concentration and specific activity in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) during metabolic studies. For measurement of specific activity, apoB was separated from other apolipoproteins and lipids by precipitation in, and subsequent washing with, isopropanol. For determination of apoB concentration, equal volumes of lipoprotein and isopropanol were mixed, and the protein content of the apoB precipitate was measured by the difference between total lipoprotein protein and the protein measured in the supernatant. Evidence that there was no apoB solubilization in isopropanol and that precipitated apoB was virtually free of soluble apolipoproteins was obtained by electrophoresis. Lipid contamination of the apoB precipitate was less than 1%. The methods were applicable to VLDL, intermediate density lipoprotein (IDL), and LDL from normolipemic patients with protein concentrations between 187 micrograms/ml and 1898 micrograms/ml. The data obtained using isopropanol were highly correlated with those using tetramethylurea, and recoveries of apoB were similar. Furthermore, the isopropanol method has been demonstrated to yield consistent data for apoB specific activities in a study of VLDL, IDL, and LDL metabolism.     

Determination of the concentration of protein by dry weight--a comparison with spectrophotometric methods

A protocol for dry weight determination of the concentration of protein, using 0.2-1.0 mg of protein per sample, has been presented and applied to nine proteins: bovine serum albumin, ovalbumin, bovine carbonic anhydrase B, galactoside binding protein (rabbit), lens calinaris lectin B, green pea lectin, soy bean agglutinin-m, wheat germ agglutinin, and antithrombin III. Dry weights, combined with spectrophotometry, have been used to calculate A1% 1 cm values at 280 nm for these proteins in dilute salt solutions and are compared with published values. From absorptivities at 288 and 280 nm in 6 M guanidine hydrochloride, the number of tryptophan residues per molecule has also been calculated and compared with literature values. These results demonstrate the utility of the present method of dry weight determination.     

Protein fractionation according to molecular size in constant pH media with immobilized charges in colinear porosity gradient

A new method of protein electrophoresis is described here. Electrophoretic separation is performed in gel media with uniform concentration of immobilized charges, combined with porosity gradient directed against protein movement. Successful separation becomes possible due to the effect of strong sample zone compression; the latter effect is connected with complex conductivity profile dynamics in a gel system containing immobilized charges. Immobilized buffers combined with porosity gradient provide an opportunity of protein discrimination based on molecular size, while in the case of uniform gel concentration the separation is based on mobility differences and strongly affected by non-uniform electric field strength profile. The proposed method does not require ionic detergent for protein separation according to their molecular weight.     

Isoelectric protein purification in segmented immobilized pH gradients. Effect of salts on rate of contaminants' removal

A method is described for keeping a constant salt background during protein purification in a segmented immobilized pH gradient. It is based on an external hydraulic flow replenishing the salt loss due to combined electric and diffusional mass transport (similar to the concept of Ribes' steady-state rheoelectrolysis). Such a minimum of ionic strength might be needed for proteins which tend to precipitate and aggregate at or in vicinity of the isoelectric point. However, it is found that any salt level in the sample feed (already at 1 mM concentration) deteriorates transport of non-isoelectric proteins, because of the much larger current fraction carried by the ions themselves as opposed to proteins. In addition, high salt levels in the sample reservoir might form cathodic and anodic ion boundaries, alkaline and acidic, respectively, which might hamper protein migration and even induce denaturation. Thus, when high salt backgrounds are needed in the sample feed, external pH control should be exerted, e.g. with a pH-stat. Three parameters influence protein transport in the segmented IPG chamber: (a) cross-sectional area of the Immobiline membranes; (b) delta pI between the isoelectric protein and the contaminants and (c) salt molarity in the sample reservoir. The first 2 show a positive, the last a negative correlation.     

Gradient reconstitution of membrane proteins for solid-state NMR studies

We here adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. We established conditions under which quantitative reconstitution of active protein at low lipid-to-protein ratios can be obtained, and also how to upscale these conditions in order to produce adequate amounts for NMR. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution, and shall represent a valuable alternative in solid-state NMR membrane protein sample preparation.     

Separation of proteins by high-speed pressure liquid chromatography

Two chromatographic systems for separation of proteins by high-speed pressure liquid chromatography are described. Molecular size exclusion chromatography was achieved by the use of porous silica deactivated by Carbowax-20M to prevent protein adsorption. Protein separations were successful provided the salt concentration in the eluting buffer was relatively high.The second system described is adsorption chromatography of proteins on deactivated Porasil. This technique involves elution of the proteins from the gel by means of a salt and pH gradient. In both systems the total time required for the chromatography is less than 1 hr.     

Spectrophotometric determination of mycelial biomass

The optical density (450 nm) of samples of homogenized fungal biomass correlated linearly with the dry weight of the biomass in the samples. As shown for broths of the filamentous microfungus Neurospora sitophila, the sensitivity of the technique depended on the extent of fragmentation of fungal hyphae during homogenization: increased fragmentation increased sensitivity. The method applied during all phases of growth, was as accurate as the conventional dry weight technique and permitted rapid and simple measurement of biomass concentration.     

The effect of sodium propionate and sodium caprylate on the fatty acid content of Trichophyton rubrum

Summary From a metabolic point of view, sodium propionate and sodium caprylate in sub-fungistatic concentrations, manifest some interesting effects upon the metabolism, fatty acid synthesis, and pigmentogenesis ofTrichophyton rubrum in its earlier growth phases.Propionate at the higher concentration and caprylate inhibit growth as measured by mycelial dry weight.The higher concentration of propionate suppressed pigment production but a yellow pigment, apparently differing from others previously described, was observed consistently in cultures with the lower concentration of the salt.Caprylate in the concentration tested resulted in a culture in which fatty acid concentration was significantly decreased, however, there appears to be no correlation between fatty acid content depression and mycelial dry weight.Data on fatty acid composition suggest that propionate, an odd-numbered carbon chain fatty acid, diverts synthesis of even-numbered carbon chain fatty acids to the odd-numbered ones.     

Further Studies on Growth and Osmoregulation of Sugar Beet Leaves under Low Salinity Conditions

In previous work, Nunes and Dias (1980) demonstrated that lowsodium concentrations in the root medium of intact or decapitatedyoung sugar beet plants grown under controlled conditions modifiedleaf water relations and increased leaf area and dry weight.The present study confirms these findings and presents furtherresults concerning the effect of salt on the concentrationsof the main osmotic substrates and on the structural and chemicalfractions of the cell dry weight. Increases of water and turgor potentials (0.25 MPa and 0.4 MPa,respectively) and a small decrease in osmotic potential (0.16MPa) were found in the leaves of salt treated plants. In theseplants, osmotic potentials estimated from the concentrationof ions and organic solutes in the leaf sap agree with thosemeasured showing that the observed increase in sodium concentrationmay account for the small decrease in the osmotic potential.No changes were detected in the concentration of orthophosphateor malic acid but total acidity of the leaf sap from salt treatedplants was significantly lower. It was found that all the main components of cell dry matter(total protein, soluble sugars, pigments and crude cell wall)contributed to the dry weight increase in the salt treated plants.Among the polysaccharide fractions of the cell wall, pectinsincreased significantly relative to hemicellulose and cellulose. Key words: Sugar beet, Sodium chloride, Growth, Osmoregulation     

The determination of density and molecular weight distributions of lipoproteins by sedimentation equilibrium.

A method is presented by which an experimental record of total concentration as a function of radial distance, obtained in a sedimentation equilibrium experiment conducted with a noninteracting mixture in the absence of a density gradient, may be analyzed to obtain the unimodal distributions of molecular weight and of partial molar volume when these vary concomitantly and continuously. Particular attention is given to the caracterization of classes of lipoproteins exhibiting Gaussian distributions of these quantities, although the analysis is applicable to other types of unimodal distribution. Equations are also formulated permitting the definition of the corresponding distributions of partial specific volume and of density. The analysis procedure is based on a method (employing Laplace transforms) developed previously, but differs from it in that it avoids the necessity of differentiating experimental results, which introduces error. The method offers certain advantages over other procedures used to characterize and compare lipoprotein samples (exhibiting unimodal distributions) with regard to the duration of the experiment, economy of the sample, and, particularly, the ability to define in principle all of the relevant distributions from one sedimentation equilibrium experiment and an external measurement of the weight average partial specific volume. These points and the steps in the analysis procedure are illustrated with experimental results obtained in the sedimentation equilibrium of a sample of human serum low density lipoprotein. The experimental parameters (such as solution density, column height, and angular velocity) used in the conduction of these experiments were selected on the basis of computer-simulated examples, which are also presented. These provide a guide for other workers interested in characterizing lipoproteins of this class.     

A fast and versatile method for extraction and quantitation of long-chain acyl-CoA esters from tissue: content of individual long-chain acyl-CoA esters in various tissues from fed rat.

A method for the extraction of acyl-CoA esters from tissue, and their subsequent analysis by HPLC is described. The lipids are removed by a two-phase extraction in a chloroform/methanol/water system. The long-chain acyl-CoA esters are extracted using methanol and a high salt concentration (2 M ammonium acetate). Reextraction of the dry residue after evaporation of extraction solvent results in low overall recoveries (20%). By adding 1 mg/ml acyl-CoA-binding protein to the extraction solvent the overall recovery was increased to 55%. The method is easy and fast to perform and is thereby suitable for analysis of a large number of samples. The advantages of the method over previously published methods are discussed.     

盐胁迫对裸果木幼苗生长和叶片相关生理指标的影响

裸果木起源于古地中海,为亚洲中部荒漠区分布的第三纪孑遗植物种,对研究旱生植物演化过程具有重要的科学价值。以一年生裸果木(Gymnocarpos przewalskii)实生苗为材料,在盆栽条件下用质量浓度为0.4%、0.8%、1.2%和1.6%的NaCl溶液进行盐胁迫处理,测定各NaCl处理下裸果木幼苗叶片的抗氧化酶活性、渗透调节物质含量、叶绿素含量、相对电导率(REC)、丙二醛(MDA)含量以及株高、基径,根、茎、叶、总干重,根冠比变化,探讨裸果木幼苗对盐胁迫的生理耐受性。结果显示,(1)随着盐胁迫程度的加重,裸果木幼苗株高、基径,以及根、茎、叶干重和总干重整体呈下降趋势,而根冠比呈上升趋势;裸果木幼苗株高、基径和根干重在0.4%NaCl处理下较对照变化不显著,但其茎干重、叶干重和总干重在各NaCl处理下均显著低于对照。(2)随着盐胁迫程度的加重,裸果木幼苗叶片的可溶性糖(SS)含量、可溶性蛋白(SP)含量、超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性均先升高后降低,而脯氨酸(Pro)含量、过氧化氢酶(CAT)活性、REC、MDA含量均呈持续升高趋势;在0.8%NaCl处理...