Isolation and characterization of hemolytic genes from Actinobacillus actinomycetemcomitans

Abstract Periodontopathic Actinobacillus actinomycetemcomitans produces hemolysin and other leukotoxins. In the present study, two distinct clones which lysed horse erythrocytes were isolated by screening genomic DNA libraries of A. actinomycetemcomitans ATCC 43718 on blood agar plates. DNA hybridization analysis indicates that there were two distinct hemolytic genes present. Sonicated extracts from both Escherichia coli clones possessed hemolytic activities on horse, sheep and human erythrocytes, but not those of rabbit. Rabbit antiserum to A. actinomycetemcomitans ATCC 43718 whole cells inhibited the hemolytic activities of these clones.     

Plasmodium berghei: characterization of antigens and their role in inducing immunity to infection

In vitro, Plasmodium berghei infected erythrocytes incorporated 35S-methionine into 31 polypeptides with molecular weights from 21 kd to 300 kd. Hemoglobin and additional smaller molecular weight polypeptides were labelled with 35S-methionine by a population of uninfected, reticulocyte-rich rat erythrocytes. 3H-glucosamine was incorporated into at least 3 components by Plasmodium berghei infected erythrocytes. Uninfected, reticulocyte-rich rat erythrocytes did not incorporate 3H-glucosamine. Rabbit antisera against small, free plasmodia formed complexes which contained between 12 and 22 of the 31 labelled polypeptides in the 35S-methionine labelled antigen preparation. Rabbit antisera against soluble antigens washed from small, free plasmodia formed complexes containing many of the same labelled plasmodial polypeptides, however the reactions were particularly strong with those components which yielded polypeptides with molecular weights of 25 kd and 31 kd. Rabbit origin antisera against the 2 preparations did not form detectable complexes with the 3H-glucosamine labelled plasmodial components. Sera from rats undergoing progressive P. berghei infection formed complexes containing an increasing number of 35S-methionine labelled plasmodial polypeptides. Hyperimmune rat serum, the only serum protective upon passive transfer into mice, formed complexes containing 7 polypeptides with molecular weights of 35 kd, 75 kd, 80 kd, 92 kd, 100 kd, 150 kd and 190 kd. Antigens containing 1 or more of these polypeptides may be important in the induction of a protective antibody response against the parasite.     

Babesia argentina: observations on the immunogenicity of the cryofibrinogen complex.

Rabbit antisera were prepared against the soluble and insoluble fractions of a cryofibrinogen complex that formed in plasma of cattle acutely infected with Babesia argentina. Analyses of the rabbit antisera indicated that the cryofibrinogen complex contained proteins from erythrocytes and parasites as well as fibrinogen and related proteins.     

Streptavidin-induced lysis of homologous biotinylated erythrocytes. Evidence against the key role of the avidin charge in complement activation via the alternative pathway

It is shown that non-covalent attachment of streptavidin, as well as of avidin, to biotinylated human erythrocytes induces homologous hemolysis by complement. Rabbit antiserum against human C3 is found to inhibit the lysis specifically as compared with non-immune rabbit serum. Efficiency of lysis inhibition is greater for avidin- and streptavidin-induced lysis of biotinylated human erythrocytes than for antibody-sensitized sheep erythrocytes. In contrast to positively charged avidin (pI 11), streptavidin is a neutral protein. Hence, hemolysis of streptavidin-carrying erythrocytes is inconsistent with the suggestion on the crucial role of avidin charge in lysis. Membrane alterations (cross-linking and clusterization of biotinylated components) induced by avidin (streptavidin) seem to be a more plausible explanation for the lysis.     

THE ACCELERATION OF SAPONIN HEMOLYSIS BY PROFLAVINE

Proflavine is a very powerful accelerator of saponin hemolysis of rabbit, human, and dog erythrocytes. Lysolecithin hemolysis, on the other hand, is inhibited. Dog erythrocytes in the presence of proflavine undergo marked changes in shape, finally becoming rods of about 13 µ in length. Rabbit and human erythrocytes are not altered in form under these conditions.     

Immunologic distinction of human muscle adenylate deaminase from the isozyme(s) in human peripheral blood cells: Implications for myoadenylate deaminase deficiency

Rabbit antisera to human muscle adenylate deaminase efficiently precipitate this enzyme from crude homogenates of muscle from man, monkey, and horse, without affecting other enzymes and proteins in the sample. They fail to react, however, with the adenylate deaminase of purified human erythrocytes, granulocytes, lymphocytes, and platelets. The exclusive presence of an antigenic determinant in the muscle adenylate deaminase suggests that this isozyme is determined by a unique gene. Since patients with myoadenylate deaminase deficiency have normal levels of the enzyme in their erythrocytes and leukocytes, this constitutes the strongest evidence at present that the deficiency state is due to an inherited gene defect.     

Isolation and characterization of rabbit H of the alternative complement pathway

Rabbit factor H, a control protein of the alternative complement pathway, was isolated from rabbit serum by polyethylene glycol precipitation, DEAE-Sephacel chromatography, and gel chromatography on Sephadex G200. The protein migrated as a single-chain polypeptide with a molecular weight of 160,000 on sodium dodecyl sulfate-gel electrophoresis with Laemmli's buffer system, but hardly migrated into the gel with Fairbanks' buffer system. Physical and chemical properties of rabbit H were similar to those of human H, except that fragments produced by limited tryptic digestion from rabbit H had different molecular sizes from those produced from human H. Significant species-specificity was observed in the functional activity of factor H; activation of the alternative complement pathway was inhibited more efficiently with homologous H than with heterologous H. In contrast, factor H inhibited the hemolysis of homologous erythrocytes less than that of heterologous erythrocytes.     

Evidence that electrofusion yield is controlled by biologically relevant membrane factors

Rabbit + rabbit and human + human combinations of erythrocyte ghost membranes were fused under the same conditions with an electric pulse. Storage at 4 degrees C of ghost membranes from both rabbit and human erythrocytes showed no change with time but storage of the erythrocytes for various periods before ghost preparation showed consistent storage-dependent changes in fusion yield.     

The phospholipid-binding protein of the reproductive tract of the bull — Effect on the removal of spermatozoal cytoplasm droplets in other species and influence of antibodies on its reactivity

The phospholipid-binding protein (PBP) isolated from bull seminal vesicle fluid removed cytoplasm droplets not only from bull, but also from ram, boar and rabbit epididymal spermatozoa. However, the presence of a protein cross-reacting with anti-PBP antisera was demonstrated by immunofluorescent staining in ram seminal vesicles and ampullae. In contrast to PBP from bull, the ram PBP-like protein did not lyse bull or ram erythrocytes. Rabbit antiserum against PBP only negligibly reduced the ability of PBP to remove cytoplasm droplets from bull epididymal spermatozoa, but it inhibited the haemolytic effect of the protein.     

Studies on the binding of staphylococcal 125I-labeled alpha-toxin to rabbit erythrocytes.

Staphylococcal alpha-toxin, a hemolytic exotoxin, can be iodinated using the lactoperoxidase method. 125 I-Labeled alpha-toxin binds to rabbit erythrocytes in an apparently irreversible and highly specific manner. The binding of 125 I-labeled alpha-toxin to erythrocytes of rabbit and human reflects the species specificity of native alpha-toxin. Binding of 125I-labeled alpha-toxin is blocked by the presence of native alpha-toxin, 127I-labeled alpha-toxin, or anti-alpha-toxin antibody. Simultaneous assays of 125I-labeled alpha-toxin binding and leakage of intracellular 86Rb+ suggest that toxin binding and membrane damage are separate, sequential functions. Both the rate and extent of binding are temperature dependent. Rabbit erythrocytes possess 5 X 10(3) binding sites/cell, while human erythrocytes possess no detectable binding sites. Treatment of rabbit erythrocytes with 125I-labeled alpha-toxin appears to decrease the number of unoccupied binding sites. Chaotropic ions can inhibit 125I-labeled alpha-toxin binding and cause bound 125I-labeled alpha-toxin to dissociate from rabbit erythrocyte membranes. Treatment of intact rabbit erythrocytes with pronase reduces both the binding capacity of the cells for 125I-labeled alpha-toxin, and the cells' sensitivity to hemolysis by native alpha-toxin. It is proposed that the primary binding site for alpha-toxin in biomembranes is a surface membrane protein.     

The decline in catalytic enzyme activity concentration with aging of the rabbit erythrocyte

Rabbit erythrocytes were separated by centrifugation on a discontinuous Percoll gradient into fractions of progressively increasing cell age to measure the in vivo decline in catalytic activity of eleven enzymes during the erythrocyte life span. Erythrocyte enzymes decline exponentially at different rates. The maximal and minimal catalytic activities (erythrocyte catalytic activity at the beginning and at the end of the erythrocyte life span), the intracellular half-life of enzymes and the daily loss of catalytic activity of total body erythrocytes were estimated.     

Immunochemical studies of organ and tumor lipids XVIII. Cytolipin R determinants in the rat erythrocyte membrane

Summary Rabbit antisera to rat lymphosarcoma contain antibodies that agglutinate trypsinized rat erythrocytes. These reactions can be specifically inhibited by cytolipin R, a ceramide tetrasaccharide isolated from rat lymphosarcoma. The agglutinin in the rabbit antisera can be absorbed with untreated erythrocytes, showing that cytolipin R determinants are present in the intact rat erythrocyte membrane. Untreated erythrocytes are able to react with antibody, but presumably the number of cytolipin R determinants necessary for agglutination becomes available only after treatment with trypsin. The anti-cytolipin R antibodies in anti-rat lymphosarcoma sera that cause hemagglutination and those that fix complement with this hapten are different, since the agglutinin can be absorbed completely without appreciable decrease in complement-fixing antibody.A preliminary report was presented at the 53rd Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., April, 1969 (1969,Fed. Proc. 28:700).     

Structural relationships between human erythrocyte sialoglycoproteins beta and gamma and abnormal sialoglycoproteins found in certain rare human erythrocyte variants lacking the Gerbich blood-group antigen(s).

The human erythrocyte membrane sialoglycoproteins beta and gamma are important for the maintenance of the discoid shape of the normal erythrocyte. In this paper we show that the human erythrocyte sialoglycoproteins beta and gamma (hereafter called beta and gamma) are structurally related. Rabbit antisera produced against purified beta and beta 1 and rendered specific to the cytoplasmic portion of these proteins also react with the cytoplasmic portion of gamma. Some human anti-Gerbich (Ge) sera react with the extracellular portion of both beta and gamma. This reactivity is shown to be directed towards a common epitope on beta and gamma. However, most anti-Ge sera do not react with beta, but react with an extracellular epitope only present on gamma. All individuals who lack the Ge antigens lack beta and gamma. In some cases abnormal sialoglycoproteins are present in the erythrocytes, and these are shown to be structurally related to beta and gamma. Rabbit antisera raised against the purified abnormal sialoglycoprotein from a Ge-negative erythrocyte type reacted with the cytoplasmic portion of both beta and gamma. Unlike normal beta and gamma, the abnormal sialoglycoproteins found in Ge-negative erythrocytes migrate as a diffuse band on SDS/polyacrylamide-gel electrophoresis. Studies using endoglycosidases suggest that the diffuse nature of these bands results from carbohydrate heterogeneity and that the abnormal sialoglycoproteins contain N-glycosidically linked oligosaccharides with repeating lactosamine units. Such polylactosamine chains are not present on normal beta or gamma.     

Multicatalytic and 26 S ubiquitin/ATP-stimulated proteases in maturing rabbit red blood cells.

Rabbit red blood cells of various ages were separated on Percoll gradients and the activities of two large cytosolic proteases were measured. Both the multicatalytic protease (MCP), assayed by hydrolysis of fluorigenic peptides, and the 26 S ubiquitin/ATP-stimulated protease, assayed by degradation of ubiquitin-lysozyme conjugates, declined 3-fold or less during maturation of rabbit reticulocytes to erythrocytes. The ability of MCP to hydrolyze three classes of peptides decreased in parallel indicating that the 20 S protease is not significantly remodeled during red blood cell maturation.     

Serological studies with the ficin-antificin system.

Rabbit antisera prepared against ficin reacted with it in a series of serologic tests. Upon immunodiffusion analysis, ficin was found to consist of at least nine antigenic components. Ficin will adsorb spontaneously to erythrocytes which can be used in passive hemagglutination tests. The enzymatic activity of ficin was not abolished by antificin sera.     

Comparative distribution of glyceraldehyde 3-phosphate dehydrogenase activity in human, guinea-pig, rabbit and mouse erythrocytes

1. Guinea-pig erythrocytes contain half the activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD) present in human cells. About 60% of their total activity is membrane-bound. 2. Rabbit erythrocytes also contain half the amount of the enzyme of human red cells. The distribution of G3PD in rabbit cells, however, is similar to that of human cells with 70% of the enzyme being membrane-bound. 3. Mouse erythrocytes contain about two-thirds of G3PD activity present in human cells. All their enzyme activity is present in membrane-free hemolysate. 4. Non-human erythrocyte membrane proteins, in addition, have relatively greater amount of band 2.1, lack band 2.2, have a more heterogenous band 3 than its human counterpart, and have overlapping bands 4.1 and 4.2.     

Characterization of staphylococcal gamma-lysin.

gamma-Lysin was purified from Staphylococcus aureus strains Smith 5R and PG23 (a toxic shock syndrome isolate) by a combination of heparin-agarose and hydroxylapatite chromatography. Both strains produced two haemolytic components, designated gamma 1 and gamma 2. Though each component was weakly haemolytic they acted synergistically to potentiate haemolysis on rabbit, sheep and human blood. Rabbit and sheep erythrocytes were more sensitive to lysis by gamma-lysin than human erythrocytes. The molecular mass of gamma 1 was 32 kDa and its pI value was 9.4. gamma 2 had a molecular mass of 36 kDa and a pI value of 9.3. While both trypsin and papain acted synergistically with gamma 2 to induce increased haemolysis, no such synergism was seen with gamma 1. Also, protease inhibitors acted to inhibit synergism between gamma 1 and gamma 2. These findings suggest that gamma 1 could be a protease.     

Exchange of labeled bicarbonate and carbon dioxide with erythrocytes suspended in an elutriator

An elutriator was used to study exchange of labeled CO2 and bicarbonate with erythrocytes. Rabbit erythrocytes were suspended by centrifugation in a stream of fluid and exposed to transient injections of an extracellular indicator (125I-albumin or 22Na+), a water indicator (3H2O), and H14CO3- and/or 14CO2. Diffusion of indicators into erythrocytes was judged by comparison of initial concentrations of diffusible and extracellular indicators in the elutriator outflow. It was possible to conduct these experiments at normal hematocrits because any carbonic anhydrase released from erythrocytes by hemolysis was washed away in the elutriator flow, and ambient pH, PO2, and PCO2 were kept constant by the inflow of fresh fluid. Equilibration of HCO3- with erythrocytes was complete during the 7- to 10-s transit time through the chamber. After this exchange was irreversibly inhibited by the anion exchange inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), addition of carbonic anhydrase (100 mg/dl) accelerated exchange, but acetazolamide (20 mg/dl) was without effect. These observations were consistent with the absence of carbonic anhydrase on the surface of the erythrocytes.     

Identification of hemoglobins within individual rhesus monkey (Macaca mulatta) erythrocytes.

Rabbit and goat antibodies to monkey adult and fetal hemoglobin were prepared and purified to apparent monospecificity. After conjugation with fluorescein isothiocyanate, the antibodies were employed to identify the hemoglobin types within individual cells in peripheral erythrocyte smears. The percentage of neonatal monkey erythrocytes containing fetal hemoglobin was found to decrease with time. The existence of adult or fetal hemoglobin in the erythrocytes appeared to be mutually exclusive.