Variability of hematocrit value, blood serum beta- and gamma-globulin level and body weight in different transferrin genotypes of common vole (Microtus arvalis, Pallas 1779) from natural population

1. Heterozygotes TfF/TfE of voles did not differ in body weight, they had higher hematocrit value (males and females), higher beta-globulin fraction (males) or higher gamma-globulin fraction (females). 2. A smaller variability of parameters studied was observed in heterozygotes than in homozygotes. Small and similar variability occurred in hematocrit value and beta-globulins while higher variability occurred in body weight and gamma-globulins. 3. In common voles the faster migrating E system of transferrin may have a higher capacity for iron transport than the F system. 4. Seasonal changes of hematocrit value in vole populations seems to result from genetical and physiological variability. 5. Sexual and seasonal changes in beta-globulin level appear to be related to changes in sexual activity of the animals during the seasons. 6. In autumn, low weight of animals and high serum gamma-globulin level, with the same physiological variability of these two parameters, suggest the synthesis of gamma-globulins with tissue (muscle) proteins. 7. Autumnal increase of gamma-globulin level seems to be very important, taking into consideration the role of the protein in water retention and its filtration from the tissues. Very high level of the protein fraction may play a role in winter high mortality of voles.     

Immunoglobulin FAB-fragments: specific activity and reactogenicity. III. Comparative study of the antigenic properties of anti-encephalitic gamma-globulin and FAB-fragments isolated from it]

Parenteral administration of antiencephalytic gamma-globulin and of the Fab-fragments, extracted from it, to rabbits stimulated the formation of specific antibodies. The maximal blood serum specific antibody level in rabbits reimmunized with Fab-fragments was much lower than that following the reimmunization of rabbits with gamma-globulin. Antibodies specific to gamma-globulin and Fab-fragments were concentrated in the gamma-globulin fraction of the immune sera and could be referred to the immunoglobulins of G class.     

Chromatographic purification of gamma-globulin from human serum on ferric oxide-agarose columns

A fast chromatographic procedure for the purification of the gamma-globulin fraction of human serum on ferric oxide-containing agarose has been developed. The presence of ferric oxide (to which gamma-globulin is absorbed) increases the rigidity of the beads. They can therefore be used at high flow rates without troublesome crosslinking. The separation has been performed on a 14-ml column and on a 1.5-ml disposable column. The yield is 87 +/- 6% and the gamma-globulin fraction is homogeneous in agarose gel electrophoresis.     

Studies on the proteolytic activity of γ-globulin preparations

1. The proteolytic activities of several gamma-globulin preparations were tested. These included sulphate-precipitated human and bovine preparations and human and bovine Cohn fraction II preparations as well as purified gamma-globulin preparations. Up to 14mg. of diffusible peptides and glycopeptides/g. of gamma-globulin was liberated after dialysis and up to 10mg. of peptides/g. after incubation and trichloroacetic acid precipitation, as products of the degradation process in incubated gamma-globulin. 2. in-Aminohexanoic acid and p-chloromercuribenzoic acid, as well as heating at 60 degrees for 40min., were shown to inhibit strongly these proteolytic activities. Streptokinase was shown to activate strongly the proteolytic activity of all the human preparations (sulphate-precipitated, Cohn fraction II, and purified gamma-globulin). 3. Two distinct pH optima were shown for human and bovine gamma-globulin preparations: one at pH8, the other at pH3.8 (the latter activity could be demonstrated only in the presence of cysteine). 4. Both (131)I-labelled human Cohn fraction II and bovine fibrinogen were attacked by a sulphate-precipitated preparation of gamma-globulin. Of the synthetic substrates tested toluene-p-sulphonyl-l-arginine methyl ester was hydrolysed by both the sulphate-precipitated and Cohn fraction II preparations, as was benzoyl-l-arginine amide at pH5, but only in the presence of cysteine. 5. These data are interpreted to indicate that at least two enzymes are present in gamma-globulin preparations, one being similar to the plasmin system, the other similar to cathepsin B.     

Response and Specificity of Antibodies for Candida albicans

Rabbit antibodies for Candida albicans, reacting in agglutination and fluorescent-antibody reactions, were present in both IgM and IgG protein fractions. The two types of immune globulins were separated from ammonium sulfate-precipitated gamma-globulin either by filtration through a column of Sephadex G-200 or by diethylaminoethyl column chromatography performed by stepwise elution with various concentrations of sodium chloride. In the fluorescent-antibody test, initial separation of the IgG fraction, prior to its conjugation with dye, proved to be essential for the high specificity of this reaction. Investigation of the specificities of the two types of antibodies revealed that the IgG was highly specific, whereas the IgM was not very specific. Each antigen fraction, extracted by various methods, demonstrated its own characteristic antibody response. Only the IgG fraction yielded serotype-specific antibody useful for detection of a serotype of C. albicans in agglutination and fluorescent-antibody tests. The results indicate the importance of IgG for specific serological reactions with the Candida species.     

Forced-flow electrophoresis of proteins and viruses

This article describes a model for forced-flow electrophoresis (FFE), considering the desired species fraction removal, other fraction removals, and outlet concentrations of all species in the system. The model predicts the necessary inlet flow rate of the retentate chamber and the rate of filtration and the voltage gradient and also provides an appropriate heat balance permitting consideration of possible heat denaturation of the species. The equipment consists of two membranes and a filter, the electric field being imposed by means of external electrodes, and two fractions are obtainable. The main discriminating factor is not the pore sizes of the filter but the relative solute ionization, which depends on the pH and the ionic strength of the buffer solution. Serum proteins (albumin, gamma-globulin) and bacteriophages (M13, MS2, phiX174) have been used to characterize the separation process.     

Some characteristics of aggregates of IgG and plasma proteins in heat-treated factor VIII concentrates

Eight batches of commercial heat-treated and one untreated factor VIII concentrate from 6 producers were analyzed for their content of IgG, IgG subclasses, IgG aggregates and the presence of other plasma proteins combined with the IgG as well as for anticomplement activity. Methods used were thin-layer gel filtration, immuno-gel filtration, spot immuno-precipitate assay in a double antibody version and an agarose plate haemolysis inhibition assay of complement fixation. The IgG content varied from 0.1-6.90 g/l. In all preparations IgG existed as monomers and aggregates. Associated with the IgG were also found, at significantly increased amounts compared to normal serum and intravenous immunoglobulin, one to four of the following plasma proteins; fibronectin, fibrinogen, von Willebrand factor antigen, Clq, albumin and IgA. Three batches from two producers had high anticomplementary activity, presumably caused by the IgG aggregates. Two of these deviated strikingly from normal human serum pools in percent distribution of IgG subclasses. It is hypothesized that these aggregates can induce side effects or cause immunological aberrations.     

Protective properties of IgM in experiments on animals]

On a model of intraperitoneal infection of albino mice the authors demonstrated a protective action of the fraction enriched with IgM and of gamma-globulin isolated from the normal human blood serum, against E. coli O111. The intensity of the protective action depended on the method, duration of administration of the preparation and also on the infective dose. Protective properties of the fraction enriched with IgM were more pronounced in comparison with the gamma-globulin preparation.     

Kinetic analysis of high affinity forms of interleukin (IL)-13 receptors: suppression of IL-13 binding by IL-2 receptor gamma chain.

Interleukin-13 (IL-13) is a pleiotropic cytokine that controls growth, differentiation, and apoptosis of immune and tumor cells. To understand the mechanisms of interaction between IL-13 and IL-13 receptors (IL-13R), and the role of the IL-2 receptor common gamma chain (gammac) in IL-13 binding and processing, we have examined IL-13 binding kinetics, dissociation/shedding, and internalization in renal cell carcinoma (RCC) cell lines. We observed a new phenomena in that the apparent rate of association, but not the dissociation, was strongly related to IL-13 concentration. We also observed cooperativity phenomena in IL-13 and IL-13R interaction in control RCC (MLneo) cells, but not in cells transfected with gammac chain (MLgammac). The number of IL-13 binding sites, the effective rate of ligand association, and the dissociation rate constants were reduced in gammac-transfected cells compared to control RCC cells. Two forms of IL-13R were detected in these cell lines, which differed in the kinetics of endocytosis and dissociation/exocytosis. Only a small fraction of bound receptors (14-24%) was rapidly internalized and the same fraction of the ligand-receptor complexes was shed and/or dissociated. The expression of gammac chain did not change any of these processes. A two independent high-affinity and moderate-affinity receptor model fit the kinetic observations in gammac-transfected cells. However, in control cells, the binding kinetics were more complicated. A mathematical model that fit a set of kinetic and steady state data in control cells was selected from a set of possible models. This best-fit model predicts that 1) two different IL-13R are expressed on the cell membrane, 2) a minor fraction of IL-13R exist as microclusters (homodimers and/or heterodimers) without exogenous IL-13, 3) high morphological complexity of the gammac-negative control cell membrane affects the cooperativity phenomena of IL-13 binding, and 4) a large number of co-receptor molecules is present, which helps keep the ligand on the cell surface for a long period of time after fast IL-13 binding and provides a negative control for ligand binding via production of the high affinity inhibitor bound to IL-13. Our data demonstrate that gammac exerts dramatic changes in the kinetic mechanisms of IL-13 binding.     

Fluorescence changes in human gamma-globulin induced by free-radical activity

Low-intensity ultraviolet irradiation was used to generate free-radical activity in lipid-free human gamma-globulin. The changes were monitored by fluorescence spectroscopy and gel filtration chromatography. The pattern and the effect of thiol compounds, free-radical scavengers and antioxidants point to a free-radical-mediated process. Initial energy uptake was probably in the region of aromatic amino acid residues, followed by complex intramolecular rearrangements. Irradiation led to the formation of molecular species and complexes whose fluorescence characteristics were different from those of native gamma-globulin and indistinguishable from those observed in inflammatory exudates.     

Critical sites for the interaction between IL-2Rgamma and JAK3 and the following signaling

JAK3 is the only known protein tyrosine kinase associating with IL-2Rgamma. This interaction is supposed to be very important to IL-2 signaling. In order to identify the critical residues for these two molecular interactions and the following signal events, various mutants of gammac and JAK3 were constructed on the basis of computer analysis. The direct interaction was determined via the yeast two-hybrid system, while the signaling was analyzed with reporter genes under the control of the c-fos, c-myc, or tnf-beta promoters, respectively. Results showed that there are two key sites on gammac involved in this interaction and the following signal transduction: the critical one is E327 via electrostatic interaction, the other is L293 via hydrophobic interaction. As to JAK3, the data indicated that Y100 is important for the interaction with gammac. These results also document that the requirement for interaction between gammac and JAK3 is different to activate different signaling pathways mediated by gammac, such as c-fos, c-myc, and JAK-STAT.     

Anti-apoptotic signaling by the interleukin-2 receptor reveals a function for cytoplasmic tyrosine residues within the common gamma (gamma c) receptor subunit

The interleukin-2 receptor (IL-2R) is composed of one affinity-modulating subunit (IL-2Ralpha) and two essential signaling subunits (IL-2Rbeta and gammac). Although most known signaling events are mediated through tyrosine residues located within IL-2Rbeta, no functions have yet been ascribed to gammac tyrosine residues. In this study, we describe a role for gammac tyrosines in anti-apoptotic signal transduction. We have shown previously that a tyrosine-deficient IL-2Rbeta chain paired with wild type gammac stimulated enhancement of bcl-2 mRNA in IL-2-dependent T cells, but it was not determined which region of the IL-2R or which pathway was activated to direct this signaling response. Here we show that up-regulation of Bcl-2 by an IL-2R lacking IL-2Rbeta tyrosine residues leads to increased cell survival after cytokine deprivation; strikingly, this survival signal does not occur in the absence of gammac tyrosine residues. These gammac-dependent signals are revealed only in the absence of IL-2Rbeta tyrosines, indicating that the IL-2R engages at least two distinct signaling pathways to regulate apoptosis and Bcl-2 expression. Mechanistically, the gammac-dependent signal requires activation of Janus kinases 1 and 3 and is sensitive to wortmannin, implicating phosphatidylinositol 3-kinase. Consistent with involvement of phosphatidylinositol 3-kinase, Akt can be activated via tyrosine residues on gammac. Thus, gammac mediates an anti-apoptotic signaling pathway through Akt which cooperates with signals from its partner chain, IL-2Rbeta.     

Characterization of the γc chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID)

X-linked severe combined immunodeficiency (X-SCID) is a rare fatal disease that is caused by mutations in the gene encoding the gammac chain. In this study, 27 unrelated Japanese patients with X-SCID were examined in terms of their genetic mutations and surface expression of the gammac chain. Among 25 patients examined, excluding two patients with large deletions, 23 different mutations were identified in the IL2RG gene, including 10 novel mutations. One patient bearing an extracellular mutation and all three of the patients bearing intracellular mutations after exon 7 expressed the gammac chain on the cell surface. Overall, 84% of patients lacked surface expression of the gammac chain leading to a diagnosis of X-SCID.     

The distribution of surface antigens during fractionation of mouse liver plasma membranes

1. Antiserum to purified mouse liver plasma membranes was prepared and the partially purified gamma-globulin antibody fraction was iodinated with (125)I. The reaction of the (125)I-labelled gamma-globulin antibody with isolated mouse liver plasma membranes was studied. 2. The gammaglobulin antibody bound specifically to mouse liver plasma membranes and there was little reaction with mouse liver intracellular membranes or with surface-membrane fractions from either rat liver or pig lymphocytes. 3. ;Light' and ;heavy' mouse liver plasma-membrane subfractions bound similar amounts of gamma-globulin antibody, and this is consistent with a surface origin for the light fraction. 5. Plasma membranes were fractionated by sequential extraction with 50mm-NaHCO(3)-Na(2)CO(3) buffer, pH10.2, containing 10mm-EDTA and aq. 33% (v/v) pyridine. The alkali-soluble and -insoluble fractions and the pyridine-soluble and -insoluble fractions all reacted with the antiserum, and the cross-reactivity among the various fractions and with the total plasma membranes was investigated. 5. The results are discussed in terms of the arrangement of the antigenic determinants within the membrane.     

Characterization of two different factors chemotactic for cancer cells from tumor tissue.

Two different factors chemotactic for cancer cells were extracted in pseudoglobulin fraction of rat ascites hepatoma transplanted tissue. After chromatography on Sephadex G-50 and CM-sephadex, these factors were separated by gel filtration on Sephadex G-100. The factor a was further fractionated by immunoadsorbent chromatography with goat antirat gamma-globulin antibody and then with rabbit antirat hemoglobin antibody; it was a protein with a molecular weight of about 78,000, resembling a chemotactic factor previously reported, and its activity was thermolabile. The previously undescribed factor b was also a protein with a molecular weight of about 14,000 and its activity was thermostable. Intradermal injection of these factors at low concentrations induced an extravascular migration of circulating tumor cells and formation of metastatic secondary tumors; and little difference in the in vivo effect between these factors was observed. It was thus assumed that the combined action of these two factors may be involved in malignant invasion.     

Study of the action of phytohemagglutinin on continuous human cells]

A study was made of the agglutinating and growth-stimulating properties of phitohemaagglutinin (PHA) preparations in experiments on continuous human cells (clone line HeLa k-41 and CaVe). PHA and PHA gamma-globulin fraction possessed poor hemagglutination properties against human erythrocytes of A, B and O groups, but expressed marked cytoagglutination properties against HeLa k-41 and CaVe cells. With high PHA doses (100 and 500 microng/ml) proliferation and mitotic indx of the culture cells was lower and the percentage of dead cells and the agglutinin titre in the preparations was greater (1 : 256). With lower PHA doses (5 and 25 microng/ml) the growth was much stronger and the percentage of dead cells lower. The agglutinin titre in the preparations decreased to 1 : 16-1 : 32. PHA gamma-globulin fraction produced the strongest growth-stimulating action with the least amount of dead cells. However, the agglutinin titre in the preparations remained high (1 : 128). A conclusion was drawn that the action of PHA preparations stimulating and inhibiting the growth of continuous human cells proved to be directly connected with the agglutinin content in the preparations, since gamma-globulin PHA fraction expressed the greatest cytoagglutinating and growth-stimulating action.     

Efficient internalization of IL-2 depends on the distal portion of the cytoplasmic tail of the IL-2R common gamma-chain and a lymphoid cell environment

The common gamma-chain (gammac), a subunit of the IL-2R, is essential for high affinity ligand binding and signal transduction due to Jak3 association to gammac. Another consequence of IL-2/IL-2R interaction is rapid receptor-mediated endocytosis of the receptor-ligand complex. In the present study, we establish that this rapid endocytosis of IL-2 in a T cell tumor line is dependent upon the cytoplasmic tail of gammac. Deletion mutants of the cytoplasmic tail mapped this activity to 9 aa of gammac, 45-54 aa distal to the transmembrane region. In contrast, ligand-independent constitutive endocytosis of gammac occurred more slowly and was dependent upon a PEST sequence in a more membrane-proximal region of the cytoplasmic tail of gammac. Thus, this receptor subunit may use distinct sorting signals for its constitutive regulation and ligand-induced endocytosis. Rapid endocytosis of IL-2 was inhibited by the tyrosine kinase inhibitor genistein, implicating a role for a signal transduction pathway in IL-2 internalization. However, one T cell line bearing a mutant gammac exhibited impaired endocytosis of IL-2, despite normal IL-2-induced Jak/STAT activation. Furthermore, inefficient endocytosis of IL-2 was noted after transfection of the COS7 epithelial cell line with the IL-2R, and further reconstitution of these cells with Jak/STAT proteins did not enhance this internalization. Collectively, these latter findings indicate that rapid endocytosis of IL-2 is dependent upon cellular signaling in lymphoid cell environment that is not solely a consequence of the presence of the Jak/STAT pathway.     

Three loops of the common gamma chain ectodomain required for the binding of interleukin-2 and interleukin-7

The common gamma chain (gammac), a subunit of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors, contributes to both cytokine binding and subsequent signal transduction. Using a model-based site-directed mutagenesis strategy, we have identified residues of the mouse gammac extracellular domain that are required for normal gammac-dependent enhancement of IL-2 and IL-7 binding. One of these sites, Tyr-103, is homologous to key ligand-interacting residues in the growth hormone and erythropoietin receptors, whereas Cys-161, Cys-210, and Gly-211 may function indirectly by maintaining the functional conformation of gammac via formation of an intramolecular disulfide bond. These two cysteines are also required for the integrity of a putative epitope recognized by TUGm2, an antagonistic monoclonal antibody that blocks gammac-dependent cytokine binding and bioactivity. These results are consistent with the involvement of three predicted loops in gammac that contribute to the binding of both IL-2 and IL-7. Mutations in these loops have also been noted in the gammac gene of patients with X-linked severe combined immunodeficiency.     

High blood triiodothyronine and the euthyroid state

A high excess of circulating T3 was observed in an euthyroid woman. Agarose gel electrophoresis of serum preincubated with 125I-T3 revealed an abnormal T3-binding in gamma-globulin zone. This binding interfered with the hormone radioimmunoassay. Immunological characterization identified this protein as an IgG-K and IgG-lambda polyclonal antibody that bound T3 but not T4. Scatchard analysis of 125I-T3 binding to the gamma-globulin fraction isolated showed a single class of binding sites with a high affinity Ka = 0.4 X 10(9) L/M and maximal binding capacity of 5.2 X 10(-9) M.     

Serum proteins of the free-ranging European bison

The paper presents the values of total protein (TP) and protein fractions in the serum of 57 European bisons from the Bia?owieza Primeval Forest, 47 of these animals being free-ranging and 10 living in enclosures. It was found that the amount of TP and amount of alpha 1- and gamma-globulin fractions are significantly lower in adult females than males. The amount of TP and gamma-globulin fraction increase significantly with age in all examined Europeans bisons, whereas the amount of alpha 1- and alpha 2-globulin fractions increase only in free-ranging animals.