Effect of radiation sterilization on the immunobiological properties of the sorbed protective fraction of Bordetella pertussis

The possibility of using radiation for the sterilization of the dried preparation of B. pertussis adsorbed protective fraction has been studied. The use of gamma irradiation in a dose of 1.5 X 10(3) J/kg has been found to permit obtaining sterile preparations while preserving their initial protective properties.     

On the possible mechanism of the protective effect of Escherichia.

A model on a HEp-2 cell culture was elaborated, permitting the study of the ability of microbes to be adsorbed an to proliferate on the surface of cells and of the mechanism of their protective effect. The ability of E. coli strains to be adsorbed and to proliferate on the surface of a cell culture was found to differ. It has been demonstrated that the protection of the cell culture from subsequent infection with virulent Shigella can be explained not only by the antagonistic activity of E. coli strains, but also by their ability to be adsorbed and to proliferate on the surface of cells. A similar mechanism of protective effect is supposed in preparations of the Colibacterin type.     

The isolation of the ribosomal fraction of Clostridium perfringens type A and an evaluation of its protective activity

A method for isolation of the ribosomal fraction (RF) from the cytoplasm of type-A C. perfringens strain BP6K was developed and its chemical and antigenic properties characterized. RF has been found to possess protective properties: two subcutaneous immunizations of mice with RF preparations adsorbed on Al(OH)3 in doses of 250 and 500 micrograms (dry weight) has ensured, on the average, the protection of 41.9% of the immunized animals from 1 DCL of type-A C. perfringens strain BP6K culture.     

The effect of repeated administration of staphylococcal immune preparations on the development of local staphylococcal infection in mice

The study is concerned with the effect of repeated administration of staphylococcal immunopreparations on the development of a suppurative-inflammatory focus in the foot of the mouse. Subcutaneous administration of large doses of the antigenic complex of the staphylococcus (ACS) obtained by aqueous extraction, antiphagin and native anatoxin failed to induce an increase in sensitivity to staphylococcus. In some cases, the extent of development of the suppurative-inflammatory focus in the mice which had been given these preparations was less than in the control; this is suggestive of their protective effect. When comparing, on this model, the ACS preparations and corpuscular vaccine produced from poorly and highly virulent strains, we observed a more pronounced protective effect in the preparations from the poorly virulents strains. The extent of oedema was greater than in the control when adsorbed anatoxin was administered. The administration of staphylococcal preparations with a therapeutical purpose after staphylococcus infection caused a significant decrease in the size and intensity of manifestation of the suppurative-inflammatory focus in the foot. The model of limb oedema enabled us to reveal the sensitizing and protective effect of the preparations under study.     

Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta

Lysosomal neuraminidase from human placenta has been obtained in its active form by association of an inactive neuraminidase polypeptide with beta-galactosidase and the protective protein. Using a specific antiserum, we have now identified a 66-kDa protein as the inactive neuraminidase polypeptide. It is specifically recognized on immunoblots only in its nonreduced state, and it coprecipitates with neuraminidase activity. The 66-kDa polypeptide is substantially glycosylated (38-kDa protein core with 7-14 N-linked oligosaccharide chains), a feature characteristic of lysosomal integral membrane proteins. Specific removal of the 66-kDa neuraminidase polypeptide from glycoprotein preparations prevents the generation of neuraminidase activity. Removal of beta-galactosidase or destruction of the protective protein also hinders the formation of active neuraminidase. Reconstitution of neuraminidase activity is observed after mixing glycoprotein preparations, depleted in different components of the beta-galactosidase-neuraminidase-protective protein complex, indicating that all three components of the complex are required for neuraminidase activity. Association of the neuraminidase polypeptide and the protective protein generates unstable neuraminidase activity, whereas association with beta-galactosidase is required for stability.     

The protective activity of preparations made from the tick-borne encephalitis virus grown using different cell cultures]

The preparations of tick-borne encephalitis (TBE) virus grown in swine embryo kidney cell culture have been shown to possess pronounced protective activity per unit of virion protein E in comparison with TBE virus preparations derived from cell culture 4647 and chick embryo cell culture. The antigenic activity of all virus preparations under study has proved to be practically the same. The role of post-translation modifications of TBE virus protein E in the manifestation of some of its biological properties is discussed.     

The interaction of slow-release immunoadjuvants with selected antigens measured in vitro

A simple, rapid in vitro binding assay for measuring the binding of protein preparations to slow release immunoadjuvants is described. This spectrophotometric method requires no unstable radiolabelled or enzyme reagents and replaces the more tedious and subjective indirect hemagglutination procedure. Therefore, it provides a potential supplement to in vivo animal work in adsorbed vaccine production. The assay was used to study the interaction or complexation behaviour of two adjuvants with tetanus toxoid.     

Association of Pseudomonas cytochrome oxidase with liposomes.

Purified Pseudomonas cytochrome oxidase has been associated with asolectin liposomes by two different methods. Firstly, the enzyme was attached to liposomic membranes by adding it to a cholate-phospholipid dispersion and subsequently dialyzing the detergent out of suspension. In the second case the enzyme was adsorbed on the preformed liposomes when added to them after the dialysis. A stimulation of the cytochrome oxidase activity approximately twenty-fold was observed by the first method. In contrast, the activation was absent in the second type of preparation, indicating that interaction between the enzyme and phospholipids is very different in the two types of vesicles. The cholate-dialysis method for reconstitution of protein-phospholipid vesicles seems to lead to rather heterogenous preparations. These can be further fractionated, not only according to their size but also to the protein/phospholipid ratio, by gel chromatography.     

Adsorption of a hydrophobic mutagen to dietary fibre from the skin and flesh of potato tubers.

One of the theories to explain the protective action of some dietary fibres against colon cancer is that certain mutagens and/or cancer promoters are adsorbed to these dietary fibres making the mutagens and/or cancer promoters less available to gut mucosal cells. The abilities of 2 contrasting cell wall preparations (dietary fibre preparations) from potato tubers to adsorb in vitro the hydrophobic mutagen, 1,8-dinitropyrene (DNP), were studied using an incubation mixture containing DNP in phosphate-buffered saline (PBS). Walls from potato skins strongly adsorbed DNP and, at the highest wall concentration tested, only a small porportion of the DNP remained in solution. In marked contrast to the skin walls, potato flesh walls adsorbed only a small proportion of the DNP. Unexpectedly, the flesh walls also caused a large increase in the proportion of DNP found in solution. When flesh walls were pre-extracted with PBS, the ability of the extracted walls to bind DNP increased. The material extracted from the flesh walls was able to maintain DNP in solution, when added to the incubation medium in the absence of cell walls. Pectic polysaccharides appear to be the soluble component responsible for maintaining the DNP in solution. Competition between soluble and insoluble fibre components may have major implications for the availability and distribution of hydrophobic mutagens in the alimentary tract.     

Demonstration of a class of proteins loosely associated with secretory granule membranes

It is shown, in this study, that rat secretory granule membrane preparations, as prepared by the method of Amsterdam et al. [(1971) J. Cell Biol. 50, 187–200], contain a protein fraction which is removed by washing in isotonic medium. This fraction contains unusually high levels of Pro, Gly and Glx, and appears to label rapidly if the rats are pulsed with [14^c] amino acids prior to removal of the glands. The fraction, which may represent specifically adsorbed secretory protein(s) or peripheral membrane protein, is significant to investigators using this model system to study secretory phenomena.     

An active-site titration method for lipases

A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.     

Citrate method of extracting antigens from Shigella sonnei. I. Comparison with other methods

The comparative characteristic of extracts obtained from Sh. sonnei by the method of Raynaud and Digeon and neurotoxin obtained by sedimentation with trichloroacetic acid from chloroform autolysates by the method of Mesrobeanu et al. is presented. Both preparations were shown to be similar in their antigenic structure and immunogenicity for mice and guinea-pigs. Nevertheless the extracts obtained by the citrate method contained considerably less protein and endotoxin and had no enterotoxic activity. This allows to recommend the citrate method for extracting protective antigens from Sh. sonnei.     

Association of Pseudomonas cytochrome oxidase with liposomes

Purified Pseudomonas cytochrome oxidase has been associated with asolectin liposomes by two different methods. Firstly, the enzyme was attached to liposomic membranes by adding it to a cholate-phospholipid dispersion and subsequently dialyzing the detergent out of suspension. In the second case the enzyme was adsorbed on the preformed liposomes when added to them after the dialysis.A stimulation of the cytochrome oxidase activity approximately twenty-fold was observed by the first method. In contrast, the activation was absent in the second type of preparation, indicating that interaction between the enzyme and phospholipids is very different in the two types of vesicles.The cholate-dialysis method for reconstitution of protein-phospholipid vesicles seems to lead to rather heterogeneous preparations. These can be further fractionated, not only according to their size but also to the protein/phospholipid ratio, by gel chromatography.     

Purification and characteristics of a protective factor from Bacillus anthracis toxin

It was found that during filtration of a sterile toxic cultural supernatant (TCS) obtained by 24 hour cultivation of the vaccinal strain through a column packed with porous glass or silochrome not only oedematic (OF) and lethal (LF), but also protective (PF) factors of toxin are adsorbed on the column. Elution of adsorbed antigens allowed for rapid concentration and purification of biologically active components of toxin from large volumes of TCS under conditions of limited proteolysis. The experimental results suggest that in 24 hour TCS and PF exists as large (87 kD) molecules as well as low molecular weight fragments whose molecular mass is of the order of 17-18 kD. The PF preparations whose molecular mass is below 68 kD possess a weak biological activity.     

Colorimetric method for the assay of heparin content in immobilized heparin preparations

The properties of the metachromatic dye toluidine blue have been utilized to determine colorimetrically the amount of heparin covalently coupled to Sepharose. The method involves monitoring the dye depletion in the supernatant at 631 nm as Toluidine blue is adsorbed onto the heparin polymer upon the beaded matrix. The procedure represents a simple assay technique which allows the direct quantitation of heparin in immobilized heparin preparations.     

Histochemical Differentiation of Chloride from Other Ions Precipitated by Silver Nitrate in Freeze-Substitution Fixation

The method is based on substitution fixation at —25° C of quickly frozen tissue with a 90% alcohol solution saturated with silver nitrate. The silver salts are photochemically reduced in the histological preparations. At this low temperature very little staining of the protein structure of the tissue takes place. Silver ions adsorbed by the tissue can be removed by treatment with a sodium nitrate solution. About 2/3 of the brown material in the histological preparations of cerebral cortex was due to the chloride in the tissue, 1/6 to the phosphate, 1/10 to an unidentified (probably organic) anion, and 1/20 to bicarbonate. When the alcoholic silver nitrate solution used for the fixation is acidified, or the sections are treated with nitric acid, the colored material consists of reduced silver chloride only. A comparison of the light absorption in histological preparations of cortex treated with neutral and with acid solutions supported the conclusion that about 2/3 of the colored material in the tissue is reduced silver chloride.     

Colorimetric assay for cellular transglutaminase

A colorimetric assay for cellular transglutaminase using 5-(biotinamido)pentylamine and polyvinylidine difluoride membranes for crude cellular preparations and purified enzyme has been developed. The biotinpentylamine substrate was incorporated into N,N-dimethylcasein by transglutaminase, the biotinylated products were adsorbed onto the membrane disks and conjugated with streptavidin-beta-galactosidase, and the absorbance resulting from the formation of p-nitrophenol from hydrolysis of p-nitrophenyl-beta-D-galactopyranoside was measured at 405 nm. The validity of the assay was established by showing a good correlation, gamma = 0.922, between the colorimetric procedure and the commonly used radiometric filter paper method for the enzyme. The procedure offers a rapid, sensitive, and nonisotopic method for the estimation of cellular transglutaminase activity in as low as 20 ng of purified guinea pig liver transglutaminase and 10 micrograms of crude fibroblast cytosol protein.     

Different properties of the lipases contained in porcine pancreatic lipase extracts as enantioselective biocatalysts

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl-Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl-Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups. The different immobilized preparations showed different properties and different response due to change in the experimental conditions. Thus, in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester [(+/-)-1] to produce the corresponding acid [2], the octyl-25KDa preparation showed the best enantioselectivity (E) value (E = 7) at pH 5 and 25 degrees C, whereas the phenyl-PPL was the most enantioselective (E = 10) at pH 5, 4 degrees C, and 10% dioxane. Using different preparations at different pHs it was possible to resolve (+/-)-2-O-butyryl-2-phenylacetic acid [(+/-)-3] with a high E value (E > 100); for example, with octadecyl-33 KDa enzyme at pH 8.     

An immunologic method of producing antigens for the treatment and prevention of leishmaniasis

Antigens isolated from lysates of L. infantum promastigotes by electroelution from polyacrylamide gels and by gel filtration, have already been proven to induce anti-Leishmania protective immunity in BALB/c mice when injected subcutaneously. At the present time, five preparations including either Leishmania antigens or anti-idiotypic reagents as vaccines can be considered as candidates for immunoprophylaxis in natural hosts of Leishmania parasites. We present a new method for producing Leishmania antigenic preparations which is of considerable interest, since it can be proposed for immunotherapy and prevention of other parasitic, bacterial, viral and perhaps, retroviral diseases.     

Chromosomal proteins of Drosophila melanogaster and an approach for their localisation on polytene chromosomes

Chromosomal proteins have been prepared from embryos of Drosophila melanogaster and separated into histone and nonhistone fractions by a procedure which completely avoids exposure to extremes of pH. These fractions have been characterised by amino acid analysis and gel electrophoresis. Antisera have been prepared against whole chromatin and against the two chromosomal protein fractions. — A new method is described for the preparation of Drosophila salivary chromosomes. This method employs microdissection techniques and completely avoids the use of acid fixatives. Preservation of fine structure in these preparations is comparable to, if not better than, that in classical acid-fixed preparations. Antisera against embryo chromatin and chromosomal protein fractions react with the salivary chromosome preparations. These reactions exhibit selectivity with different chromosomal structures. Evidence is presented suggesting a specific distribution of protein antigens along the chromosome.