Methods and reagents: Glowing blue gels and nuked nucleases

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagents, available on the internet. This month's column discusses some unidentified blue fluorescence observed emanating from agarose gels, and the use of microwave ovens for DNA restriction digests. For details on how to partake in the news-group, see the accompanying box.     

ParaSite: The Pick of Parasites on the Web

While a steady flow of questions were put to the general parasitology newsgroup during November, none provoked discussion. Other discussion lists came to life:     

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand

An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.     

MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN AND MICROSOMAL FRACTIONS OF BOVINE BRAIN

Abstract— The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3',5'-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2 m -NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 m -NaCl for the microsomal enzyme. A peak with protamine and casein as substrate for the microsomal or myelin enzyme, respectively, was larger than that with histone as substrate for each enzyme. The first peak with histone as substrate on a DEAE–cellulose column appeared as two peaks on the Sepharose 6B column. The second peak with histone as substrate on DEAE–cellulose column was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and subunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6B column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size.
The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.     

Factor H is a dermatan sulfate-binding protein: identification of a dermatan sulfate-mediated protease that cleaves factor H

Dermatan sulfate mediates the blood coagulation cascade by binding to heparin cofactor II and potentiating the antithrombin activity. In order to explore another function of dermatan sulfate, a dermatan sulfate affinity column was prepared from biotinylated dermatan sulfate and Streptavidin Sepharose. When human plasma was applied on the dermatan sulfate column, factor H was bound and cleaved. The cleavage products, a 30-kDa N-terminal fragment and a 120-kDa fragment, were eluted from the column with 500 mM NaCl and detected after Western blotting with anti-factor H. The bond between the tandem arginine residues in the sixth domain of factor H was cleaved. When purified factor H was applied on the column, the factor H was not cleaved and was recovered from the column as an intact 150-kDa fraction. The finding that dermatan sulfate-mediated cleavage of factor H was inhibited by (p-amidinophenyl) methanesulfonyl fluoride, but not N-ethylmaleimide or EDTA, indicates that a serine protease in the plasma was activated on the dermatan sulfate column and factor H was cleaved without intervention of the plasma protease inhibitors. Amidase activity was detected in the effluent from the dermatan sulfate column but was abolished by pretreatment of the plasma with dermatan sulfate. Therefore, dermatan sulfate participates in the activation of a protease as well as having the protease inhibitory action.     

A system for coupled multiple-column separation of proteins

Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time.     

Effects of nonuniform column packing in analytical gel chromatography. II. Associating solutes

Effects of nonuniform column packing on boundary profiles for selfassociating systems have been investigated by computer simulation. Migration rate of each of the interconverting solute species changes along the column as a result of nonuniform packing, and the difference in velocity of monomer and n-mer is not constant as the sample moves down the column. A greater amount of overall axial dispersion results, as compared to the constant-column case. Procedures developed in this study can be applied to any experimentally measured column nonuniformity.     

Pulmonary surfactant will secure free airflow through a narrow tube

Well functioning pulmonary surfactant is necessary to ensure alveolar stability. It is proposed that surfactant is also required to keep the finest cylindrical airways open, thereby securing an unrestricted flow of air to and from the alveoli. If the surfactant is inadequate in quality or quality there is a risk that liquid will accumulate in the most marrow section of the airway and form a blocking column. To study that possibility special glass capillaries were used. The glass capillaries were heated and extended to make a short section very narrow. In the lumen of that section a minute volume (1 microliter) of liquid was deposited, which formed a blocking column. When pressure was raised on one side of the column, it forced the liquid to move away from the narrow section. Pressure dropped to zero as air could pass, and if the liquid column consisted of calf lung surfactant extract (CLSE), pressure remained at zero because a new liquid column did not form. If, on the other hand, the liquid column consisted of saline solution it would repeatedly reform as soon as it had been pressed out of the capillary's narrow section. The same occurred if the CLSE suspension forming the liquid column was very dilute or contained inhibiting proteins. These observations did not require that the capillary consisted of the material glass; they were also noted when the narrow tube was outlined by epithelium.     

Virtual resource development in the glycosciences

The development of Internet-based virtual resources is a relatively new area of scientific and technical activity that is currently undergoing rapid expansion. Major factors fuelling recent growth include the emergence of multimedia capabilities through the rapid evolution of the World Wide Web, the reduction in cost of high quality personal computers and graphics workstations and the provision of mass-marketed provider services. Prior to 1995 the presence of Internet resources in the glycosciences was virtually non-existent. Existing scientific knowledge was primarily made available on the Net through the provision of databases from gopher and ftp sites. A particular example in the glycosciences is the Carbbank database of biological carbohydrate sequences. We will describe here our efforts in 1994–95 in establishing The Glycoscience Network (TGN, http://bellatrix.pcl.ox.ac.uk/TGN/). These activities included the establishment of a newsgroup, mailing lists, Web resources and the running of the First Electronic Glycoscience Conference (EGC-1, http://bellatrix.pcl.ox.ac.uk/egc/). EGC-1 included many novel initiatives in the glycosciences including electronic posters and papers, a Virtual Conference Centre, a Web-based hyperglossary, Virtual Trade and Employment Centres, refereed electronic publishing, and the creation of a Virtual Reality Gallery. We would like to look towards the near future and discuss several initiatives in virtual resource creation that we believe will have significant scientific impact on the glycosciences including the development of bioinformatics-based servers, sophisticated interactive databases, and videoconferencing. Furthermore, we cherish the belief that these resources will foster international scientific collaboration and progress of an extent never previously possible. Finally, we indulge in speculation and make some suggestions on the form and long-term impact of Glycoscience Virtual Resources. We predict that their development may completely reconstruct the scientific environment that we work in as scientists and we reflect on the probable benefits and pitfalls to be encountered.This paper was presented at the First Electronic Glycoscience Conference (EGCI) on the World Wide Web, September 1995.     

花卉柱式无土栽培

于玻璃温室内的160 m2面积上,以干湿交替型盆钵组装起高200 cm, 直径15 cm的立柱77根,然后组成立柱“树林”并种花,25个科的共53种草花用同一营养系统管理,生长都良好,立柱“树林”象是花的“森林”,显示了良好的生态关系。在此基础上挑选不同种盆花组成不同情趣的家庭阳台花柱。阳台花柱具若干优点：新型盆钵具良好的水、气、肥协调关系,适合多种花卉生长;柱高任选,便手提携带;柱体能环绕中轴旋转使植物受光均匀;花柱底部是具中岛结构式底盆,起蓄水和稳定作用,还便于多根立柱串联扩大栽培量;有人工和自动浇灌两种系统,省工省时且干净卫生。     

Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms

Two thermostable enzymes synthesized by thermophilic microorganisms were isolated and purified. A thermostable ß-galactosidase was produced in a continuous fermentation process by Bacillus stearothermophilus TP 32 as an intracellular enzyme. After applying different concentration procedures the raw extract enzyme was prepurified on a Sephadex G-200 size exclusion column. The isolated ß-galactosidase fraction was then separated with HPLC on a TSK G 3000 SW size exclusion column to determine the molecular mass based on calibration curves of standard proteins. The other enzyme, a thermostable protease, was synthesized by Bacillus stearothermophilus TP 26 as an extracellular enzyme. After its concentration, the enzyme was purified on a classical size exclusion column (Sephacryl S-200) and on a HPLC size exclusion column (BIO-SIL TSK-250). The micropreparatively isolated fraction was separated again on this HPLC column to determine its molecular mass. The optimum temperature of both enzymes was approximately 75°C.     

The azure dyes: their purification and physicochemical properties. II. Purification of azure B.

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or polychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polymeric adsorbent (XAD-2) the acetate-formate anions are exchanged in chloride. Regeneration of both columns is possible: KMnO4, Na2S2O4 and water are run through column A; 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

Pore network modelling of affinity chromatography: determination of the dynamic profiles of the pore diffusivity of beta-galactosidase and its effect on column performance as the loading of beta-galactosidase onto anti-beta-galactosidase varies with time.

A three-dimensional pore network model for diffusion in porous adsorbent particles was employed in a dynamic adsorption model that simulates the adsorption of a solute in porous particles packed in a chromatographic column. The solution of the combined model yielded the dynamic profiles of the pore diffusion coefficient of beta-galactosidase along the radius of porous adsorbent particles and along the length of the column as the loading of beta-galactosidase onto anti-beta-galactosidase immobilized on the surface of the pores of the particles occurred, and, the dynamic adsorptive capacity of the chromatographic column as a function of the design and operational parameters of the chromatographic system. It was found that for a given column length the dynamic profiles of the pore diffusion coefficient were influenced by (a) the superficial fluid velocity in the column, (b) the diameter of the adsorbent particles, and (c) the pore connectivity of the porous structure of the adsorbent particles. The effect of the magnitude of the pore connectivity on the dynamic profiles of the pore diffusion coefficient of beta-galactosidase increased as the diameter of the adsorbent particles and the superficial fluid velocity in the column increased. The dynamic adsorptive capacity of the column increased as (i) the particle diameter and the superficial fluid velocity in the column decreased, and (ii) the column length and the pore connectivity increased. In preparative affinity chromatography, it is desirable to obtain high throughputs within acceptable pressure gradients, and this may require the employment of larger diameter adsorbent particles. In such a case, longer column lengths satisfying acceptable pressure gradients with adsorbent particles having higher pore connectivity values could provide high dynamic adsorptive capacities. An alternative chromatographic system could be comprised of a long column packed with large particles which have fractal pores (fractal particles) that have high pore connectivities and which allow high intraparticle diffusional and convective flow mass transfer rates providing high throughputs and high dynamic adsorptive capacities. If large scale monoliths could be made to be reproducible and operationally stable, they could also offer an alternative mode of operation that could provide high throughputs and high dynamic adsorptive capacities.     

The Azure Dyes their Purification and Physicochemical properties. II. Purification of Azure B

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or plychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polyineric adsorbent (XAD-2) the acetate-formate anions are exchanged for chloride. Regeneration of both columns is possible: KMnO₄, Na₂S₂O₄ and water are run through column A, 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

Enantiomeric analysis of amino acids by using comprehensive two-dimensional gas chromatography

The chiral separation of amino acids (AA) derivatised with ethyl chloroformate by using comprehensive two-dimensional gas chromatography is reported. A commercially available enantioselective capillary column (Chirasil-l-Val) has been tested as first-dimension column. Two nonenantioselective stationary phases (BPX50 and BP1) with different column lengths were combined with the enantioselective column, which represent chiral/polar and chiral/low-polarity column sets, respectively. These column sets were evaluated to determine the most useful column combination to provide improved separation efficiency of enantioselective AA analysis. Separations of AA mixtures derivatised either as their N-trifluoroacetyl methyl esters or with methyl chloroformate, performed on a chiral/low-polarity column set, are also shown. The method was demonstrated for chiral analysis of AAs in different beer samples. The major AA in the beer samples was proline with amounts ranging from around 65-95% with minor contents of glycine and the l-enantiomers of alanine, valine, leucine, and isoleucine. Small amounts of d-alanine, at about 1, 1.5, and 15% were detected in the three samples.     

Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column

This study examined the impact of fouling with yeast homogenate on capacity and breakthrough performance of an ion exchange packed bed column. Column performance was assessed by analysis of breakthrough curves obtained with BSA as a test protein. The overall impact of fouling on breakthrough performance depended heavily on the level of clarification of the feed stream. Challenging the column with particulate-free homogenate caused no change in column performance. Loading successive small volumes of poorly clarified homogenate, interspersed with frequent column salt washes, did not alter significantly the column capacity. By contrast, when the column was challenged with an equivalent cumulative volume of poorly clarified homogenate, dynamic binding capacity decreased significantly and changes in breakthrough curves suggested increased intraparticle and external mass transfer limitations. These changes were ascribed to deposition of solid particulates in void spaces in the bed and colloidal contaminants in the bead pores.     

Efficacy of a pentaiodide resin disinfectant on Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocysts in vitro

The resin-I5 column developed at Kansas State University was tested for efficacy against oocysts of Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae). Cesium chloride gradient-purified oocysts were passed through 1.0-cm-diameter columns with lengths of 2.5, 5.0, and 10.0 cm at 23 C. Following column passage, oocyst viability was determined both in vitro by excystation and in vivo by the ability to establish infections in suckling mice. Oocysts were found to be retained by the pentaiodide resin in a linear fashion, probably by electrostatic interactions. Linear regression analysis revealed 100% of the oocysts should be removed in such a manner using a column length of greater than or equal to 25.7 cm. When compared to untreated control oocysts, less than 12% of the oocysts that passed through the columns appeared to be affected by the resin, as assessed by excystation. Inoculation of suckling mice with these column-treated oocysts supported the excystation data and revealed the coccidian to be viable. These results indicate that oocysts of C. parvum are retained on the pentaiodide column in a 1-hit manner and that, although killing of parasites may occur within the column, the greatest effect that the column may have on the parasite is as an electrostatic retention device.     

Affinity chromatography of protein kinase C-phorbol ester receptor on polyacrylamide-immobilized phosphatidylserine

An affinity column, prepared by immobilizing phosphatidylserine and cholesterol in polyacrylamide, was utilized in the purification of protein kinase C. Protein kinase activity and phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylation of histone H1 and [3H]phorbol dibutyrate binding, respectively. Both activities were present in a cytosolic extract of rabbit renal cortex, eluted together from a DEAE-cellulose column, bound to the affinity column in the presence of Ca2+, and eluted symmetrically upon application of EGTA. Recovery from the affinity column was high (30-50%) and resulted in as much as a 6000-7700-fold purification, depending on the region of the DEAE-cellulose peak that was applied. Following affinity column purification, protein kinase and phorbol ester binding activity eluted symmetrically upon gel filtration, with a molecular weight of approximately 80 kDa. A protein of the same size was present in silver-stained gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity column purified samples from the DEAE-cellulose peak. From 2-4 other, smaller proteins were also present, their number and relative amounts depending on the region of the DEAE-cellulose peak used. These data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2+ plus phospholipid-dependent interaction.     

Prediction of Catalytic Residues Using the Variation of Stereochemical Properties

In this paper, we investigate a simple protein sequence conservation measure which takes amino acid similarity into account. Instead of grouping 20 amino acids into disjoint sets in previous methods, we consider ten overlapping classes. The method is based on the assumption that a column in a multiple sequence alignment is evolved from an identical column in the evolutionary history. Two ten-dimensional vectors are constructed for each position to denote frequencies of ten classes in a column and the corresponding hypothetical identical column. Then the cosine function of the angle between these two vectors is considered as a measure of divergence of stereochemical properties at this position. This divergence, combining with other conservation scores, is used as conservation measure of the column. Finally, we evaluate our methods by identifying catalytic sites, using rank analysis criterion and receiver operator characteristic analysis criterion.     

A model for feature linking via collective oscillations in the primary visual cortex

A neural network model for explaining experimentally observed neuronal responses in cat primary visual cortex is proposed. In our model, the basic functional unit is an orientation column which is represented by a large homogeneous population of neurons modeled as integrate-and-fire type excitable elements. The orientation column exhibits spontaneous collective oscillations in activity in response to suitable visual stimuli. Such oscillations are caused by mutual synchronization among the neurons within the column. Numerical simulation for various stimulus patterns shows that as a result of activity correlations between different columns, the amplitude and the phase of the oscillation in each column depend strongly on the global feature of the stimulus pattern. These results satisfactorily account for experimental observations.