Methodological studies on the immunologic determination of proconvertin (factor VII)]

The influence of the different determinations of factor VII activities on the determination of the concentrations of the immunoreactive factor VII in the neutralizing test according to Good-Night were re-examined. The measurement of activity was performed with Seitz filtered cattle plasma as a complex proof of factor VII and X and with a specific artificial deficiency plasma. The examinations had the following results: In the plasma of healthy grown-up people there is a considerable, apparently physiologic range of dispersion of the immunoreactive concentration of factor VII within its normal activity which can be traced irrespective of the used measurements of activity. In plasma samples with a decreased activity of factor VII the values of measurement of the complex evidence will surpass those of the specific determination. The extent of neutralizing the activity in factor VII is not only dependent on the antibody concentration, but will mostly depend on the antibody-antigen relation. The findings obtained prove that the natural deficiency plasma of factor VII used in the original method of Goodnight can be replaced by an artificial substrate plasma.     

An improved method for purification of L-threonine deaminase from rat liver

L-threonine deaminase was obtained at a high degree of purity from rat liver. Two main steps were added to the previously reported procedure: cryoprecipitation and chromatofocusing (in the presence of a specific KCl concentration). The purification factor was 3,090 and the specific activity 989. The method is very reproducible and convenient. It gives the highest specific activity and the highest degree of purity of the enzyme recorded to date.     

Clones of the lymphoblastoid line RPMI-6410t requiring an exogenous growth factor

It was earlier established that the RPMI-6410t cells, obtained from a patient with acute myeloblastemia, synthesized a growth factor which maintains their proliferation and had a specific receptor for this factor on their surface. The use of a medium conditioned by the 6410t cells made it possible to define conditions in which practically 100% efficiency cloning of these cells is attained by the method of limiting dilutions. In the present work, this method of cloning was applied to obtain from the 6410t strain clones which are characterized by a requirement for an exogenous growth factor. These clones, like the 6410t cells, have on their surface specific receptors but, unlike the parental cells, do not synthesize the growth factor and do not form colonies in soft agar, i. e. lose one of the features of malignancy. These facts agree with the published data according to which the proliferation of normal cells is regulated by exogenous growth factors and confirm a suggestion put forward in our previous work that the endocrine regulation of cell growth is one of the mechanisms of malignancy.     

Thromboplastin immobilized on polystyrene surface exhibits kinetic characteristics close to those for the native protein and activates <Emphasis Type="Italic">in vitro</Emphasis> blood coagulation similarly to thromboplastin on fibroblasts

A method for transmembrane protein thromboplastin (tissue factor) immobilization on polystyrene surface is described. Tissue factor is the main activating factor launching the blood coagulation process. It is a cofactor of factor VIIa, the first protease in the cascade of coagulation reactions. The proposed method preserves kinetic characteristics specific for native tissue factor on the fibroblast surface. The kinetics of binding to factor VIIa and enzymic activity of the formed complex follow Michaelis-Menten kinetics, which is also characteristic of native complex. A small difference is that dissociation constant for tissue factor immobilized on polystyrene surface exceeds 2.7-fold that for native factor. The proposed technique of immobilization provides for protein density on the activating surface corresponding to the tissue factor density on the fibroblast surface. The immobilized tissue factor can be used to activate blood coagulation in methods simulating spatial dynamics of in vitro clot growth. Investigation in this direction will make it possible to register both hypo- and hypercoagulation states of the system. This approach is advantageous over traditional methods of estimation of the coagulation system conditions, which mainly register only hypocoagulation. Investigation of the storage time has shown that activators containing immobilized tissue factor can be stored and used during for at least 100 days in the method studying spatial dynamics of fibrin clot formation.     

Escherichia coli initiation factors: isolation of an IF2-fMet-transfer RNA complex

A method is described for the isolation of a stable specific complex between initiation factor IF2 and IMet-tRNA.     

Development of Dual Ensemble Monte Carlo Program and its Application to the CO2/N2 Separation

Abstract

A novel Monte Carlo simulation named as Dual Ensemble Monte Carlo (DEMC) method is developed for the investigation of the membrane separation process. In this method the spatial combination of Grand Canonical MC and Canonical MC techniques is employed. The DEMC method can be used to calculate the separation factor at a specific chemical potential gradient. At first, a check on the accuracy of the DEMC method is made by generating gas density gradient between two reservoir regions. Thereafter, we applied this method to CO₂/N₂ gas separation by inorganic membranes and calculated the separation factor dependence on the size of micropore in membranes.     

Affinity purification of aminoacyl-tRNA

A procedure for separating Escherichia coli aminoacyl-tRNA from unacylated tRNA or components of the aminoacylation reaction, thereby achieving an aminoacyl-tRNA product with a very high specific activity, is described. The method utilizes the specific recognition of aminoacyl-tRNA for E. coli protein synthesis elongation factor Tu which has been immobilized on an affinity matrix. The application of the affinity procedure as a means of purifying a single aminoacyl-tRNA from an unfractionated mixture of tRNAs is also discussed.     

A chemical method to enrich RNA by molecules having 5''-terminal triphosphate groups

A method is proposed to enrich RNA molecules having a 5'-terminal triphosphate group. The method is based upon selective chemical modification of 5'-triphosphate groups by an antigen-containing amine followed by affinity chromatography on an absorbent loaded with antibodies specific to this antigen. A purification factor up to 37 may be achieved.     

Simultaneous detection of multiple single-base alleles at a polymorphic site.

PCR amplification of multiple specific alleles (PAMSA) is a rapid method of detecting single-base polymorphisms. We demonstrate its utility by detecting a polymorphism in an Alu sequence in the factor IX gene.     

Complement proteins and macrophages. 1. Quantitative estimation of factor B produced by mouse peritoneal macrophages

Cobra venom factor was used for the detection of factor B synthesized by mouse peritoneal macrophages. This method was shown to be specific for factor B assay by neutralization by antimouse factor B antibody. The amount of factor B in the culture supernatant, assessed by this method, was found to be dependent on the medium used for cultivation of macrophages. The addition of 25% L cell-conditioned medium to minimal essential medium (LCM-MEM) enhanced the production of factor B and also of lysozyme. Kinetic analysis in LCM-MEM showed that factor B produced by 6 x 10(4) cells/cm2 increased up to 72 hr and reached a plateau at 96 hr. The amounts of factor B and lysozyme produced in LCM-MEM depended upon the number of macrophages. Production of factor B was completely inhibited by 1 microgram of cycloheximide per ml and was restored by its removal.     

Rapid colorimetric quantification of lipo-chitooligosaccharides from Mesorhizobium loti and Sinorhizobium meliloti

Nod factors are lipids with a chitinlike headgroup produced by gram-negative Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are essential signaling molecules for accomplishing symbiosis between the bacteria and roots of legume plants. Despite their important role in the Rhizobium-legume interaction, no fast and sensitive Nod factor quantification methods exist. Here, we report two different quantification methods. The first is based on the enzymatic hydrolysis of Nod factors to release N-acetylglucosamine (GlcNAc), which can subsequently be quantified. It is shown that the degrading enzyme, glusulase, releases exactly two GlcNAc units per pentameric nodulation factor from Mesorhizobium loti factor, allowing quantification of LCOs from Mesorhizobium loti. The second method is based on a specific type of Nod factors that are sulfated on the reducing GlcNAc, allowing quantification analogous to the quantification of sulfolipids. Here, a two-phase extraction method is used in the presence of methylene blue, which specifically forms an ion pair with sulfated lipids. The blue ion pair partitions into the organic phase, after which the methylene blue signal can be quantified. To enable Nod factor quantification with this method, the organic phase was modified and the partitioning was evaluated using fluorescent and radiolabeled sulfated Nod factors. It is shown that sulfated LCOs can be quantified with this method, using sodium dodecyl sulfate for calibration. Both methods allow Nod factor quantification in parallel enabling a fast and easy detection of nanomole quantities of Nod factors. Accurate Nod factor quantification will be crucial for characterization and cross-comparison of the affinity for Nod factors of newly identified Nod factor binding proteins or putative Nod factor receptors.     

Detection of human factor B biosynthesis in culture supernatants and cell lysates by ELISA

We report here on the development of a simple, sensitive, convenient, and quantitative enzyme-linked immunosorbent assay for human factor B, a protein of the alternative complement pathway, by the sandwich method using goat anti-human factor B antibody. The assay described herein is reproducible and highly specific for human factor B. The assay was used to determine biosynthesis (cell lysates or extracts) and secretion (supernatants) of human factor B using human monocyte cell line U937. Phorbol myristate acetate strongly enhanced (10- to 20-fold) biosynthesis of factor B by U937. The combination of a sensitive enzyme-linked immunosorbent assay and phorbol myristate acetate enabled us to use a microculture system.