Effect of beta-endorphin and myelopeptides on the cAMP level and proliferation of lymphocytes in vitro

Beta-endorphin (10(-11)-10(-9) M) has been shown to induce naloxone-independent depression of the proliferative activity of human peripheral lymphocytes (HL), stimulated by pokeweed mitogen without affecting PHA-stimulated HL proliferation. Beta-endorphin (10(-10)-10(-7) M) also caused changes in HL cAMP level, that were blocked by naloxone. Marked individual sensitivity to beta-endorphin effects has been noted. It has been also shown that a bone marrow preparation, stimulating antibody production (myelopeptides), causes naloxone-independent depression in the proliferative activity of HL, stimulated by PHA and pokeweed mitogen, as well as naloxone-blocked decrease in cAMP HL level. It has been concluded that beta-endorphin interacts with several types of opiate lymphocyte receptors and that opioids, contained in myelopeptides, are involved in the realization of myelopeptide effect on lymphocytes.     

Study of antibody/antigen binding kinetics by total internal reflection ellipsometry

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.     

Myelopeptide correction of the differentiation of hematopoietic precursor cells in mice with experimental T-immunodeficiency

The influence of myelopeptides on differentiation of bone marrow haemopoietic precursors cells in thymectomized and normal mice has been studied in vivo. The introduction of myelopeptides decreased the number of erythroid (E) colonies and increased that of granulocytic ones (G). This results in the decrease of initially raised E/G ratio in thymectomized mice (from 4.3) down to 1.3). Myelopeptides exerted no influence on haemopoietic precursors in normal mice (E/G-2.0).     

The protective properties of myelopeptides in the development of infectious processes caused by bacteria of the genus Salmonella

The protective properties of myelopeptides in the development of bacterial infection in mice and young pigs, caused by S. typhimurium 415, S. cholerae-suis 1422 and 370, have been studied. Myelopeptides have been found to possess protective properties when injected into animals infected with S. typhimurium and S. cholerae-suis in lethal doses. The best protective effect (survival rate of 100%) has been achieved by the injection of myelopeptides 24 hours before challenge. Myelopeptides have also been found to promote the weight gain of young pigs infected with S. cholerae-suis.     

Immobilized isocrite dehydrogenase: some aspects of its catalytic, chemical and immunological properties

Isocitrate dehydrogenase from Azotobacter vinelandii has been immobilized on Sepharose 4B with an efficiency of between 60 and 75%. The immobilized enzyme is assayed by a flow technique which monitors a final steady state level of product formation. By the assay system described it is estimated that the immobilized enzyme retains between 30 and 40% of the catalytic activity of the free enzyme. Studies have been carried out on the substrate dependence of the enzyme. The enzyme requires magnesium ions with optimal concentrations of 10⁻³m and above. The dependence on isocitrate and TPN⁺ concentrations was determined and analyzed by double-reciprocal plots. The immobilized enzyme is inactivated by DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] and reactivated by DTT (dithiothreitol). The DTNB-modified enzyme can be reactivated by potassium cyanide. Comparison of these reactions with those of the free enzyme suggest that the steric environment of the active site was not grossly altered by immobilization. Some supporting evidence is derived from the identity of the energies of activation, 16,600 cal/mole, of free and immobilized enzyme catalyzed oxidation of isocitrate. Furthermore, the immobilized enzyme is inactivated by antibody prepared against the free enzyme. The covalently attached enzyme is resistant to tryptic digestion except in the presence of 2 m urea. This suggests that exposed lysyl residues which may be the primary site of attack by trypsin are utilized in immobilization. Treatment of the enzyme with 2 m urea unfolds the enzyme to a conformation which has very little activity but which recovers full activity upon removal of the urea. Interaction of the enzyme with antibody suggest that the antibody reacts univalently. The second valence can be satisfied by addition of free enzyme. The free enzyme bound to the immobilized enzyme-antibody complex is active. Preliminary attempts to dissociate the enzyme-antibody complexes have been unsuccessful.     

Changes in monoclonal antibody productivity of recombinant BHK cells immobilized in collagen gel particles

Animal cell perfusion high density culture is often adopted for the production of biologicals in industry. In high density culture sometimes the productivity of biologicals has been found to be enhanced. Especially in immobilized animal cell culture, significant increase in the productivity has been reported. We have found that the specific monoclonal antibody (MAb) productivity of an immobilized hybridoma cell is enhanced more than double. Several examples of enhancing productivities have been also shown by collagen immobilized cells. Immobilized cells involve some different points from non-immobilized cells in high density culture: In immobilized culture, some cells are contacted together, resulting in locally much higher cell concentration more than 10⁸ cells/ml. Information originating from a cell can be easily transduced to the others in immobilized culture because the distance between cells is much nearer. Here we have performed collagen gel immobilized culture of recombinant BHK cells which produce a human IgG monoclonal antibody in a protein-free medium for more than three months. In this high density culture a stabilized monoclonal antibody production was found with around 8 times higher specific monoclonal antibody productivity compared with that in a batch serum containing culture. No higher MAb productivity was observed using a conditioned medium which was obtained from the high density culture, indicating that no components secreted from the immobilized cells work for enhancing monoclonal antibody production. The MAb productivity by the non-immobilized cells obtained by dissolving collagen using a collagenase gradually decreased and returned to the original level in the batch culture using a fresh medium. This suggests that the direct contact of the cells or a very close distance between the cells has something to do with the enhancement of the MAb productivity, and the higher productivity is kept for a while in each cell after they are drawn apart.     

Feasibility of protein A for the oriented immobilization of immunoglobulin on silicon surface for a biosensor with imaging ellipsometry

The feasibility of using protein A to immobilize antibody on silicon surface for a biosensor with imaging ellipsometry was presented in this study. The amount of human IgG bound with anti-IgG immobilized by the protein A on silicon surface was much more than that bound with anti-IgG immobilized by physical adsorption. The result indicated that the protein A could be used to immobilize antibody molecules in a highly oriented manner and maintain antibody molecular functional configuration on the silicon surface. High reproducibility of the amount of antibody immobilization and homogenous antibody adsorption layer on surfaces could be obtained by this immobilization method. Imaging ellipsometry has been proven to be a fast and reliable detection method and sensitive enough to detect small changes in a molecular monolayer level. The combination of imaging ellipsometry and surface modification with protein A has the potential to be further developed into an efficient immunoassay protein chip.     

Effect of bone marrow mediator myelopeptides on the summation-threshold index and behavioral reactions of rats

Transmitter peptides having immunostimulant and endorphine-like properties were isolated from supernatant of medullary cell culture. Bioregulatory peptides were called myelopeptides. Myelopeptides provoked changes of the summation-threshold index in rats, which augmented in time. These changes pointed to the realization of the analgetic effect of myelopeptides via the spinal-stem structures of the central nervous system. Having a remarkable analgesic effect myelopeptides administered in the doses tested did not produce any action on the behavioral responses. The latter circumstance makes them differ from narcotic analgesics and known endorphines.     

Supramolecular formation of antibodies with viologen dimers: utilization for amplification of methyl viologen detection signals in surface plasmon resonance sensor

Monoclonal antibodies for 1-(carboxypentyl)-1'-methyl-4,4'-bipyridinium dichloride have been prepared. The complex formation of one of the antibodies, 10D5, with viologen dimer has been studied by a biosensor technique based on surface plasmon resonance. The dissociation constants of the complex between antibody 10D5 and methyl viologen or viologen dimer are found to be (2.0 +/- 0.2) x 10 (-7) and (1.5 +/- 0.5) x 10 (-7) M, respectively. Enhancement of response signal intensities in SPR is observed by the addition of the antibody solution to the viologen dimer-antibody complex indicating the formation of linear supramolecules between the antibody and viologen dimer. Amplification of methyl viologen sensing processes is realized by the inhibition of the complex formation between antibodies and viologen dimer-antibody complexes by methyl viologen and signal enhancement due to the supramolecular formation of the antibody and viologen dimer. The sensitivity in this system is found to be 2 orders larger than that obtained in the simple addition of methyl viologen to the antibody immobilized to the surface of the sensor chip.     

Effect of B-activin on the rosette-forming capacity of mouse lymphocytes at the peak of a secondary immune response to ram erythrocytes

The effect of B-activin secreted by bone-marrow cells in response to the expression of antigen-recognizing receptors at the peak of immune response to SRBC was studied. A negative correlation between the number of immune RFC and antibody-producing cells under the influence of B-activin was established. It was shown that the decrease in the number of immune RFC was the result of precommitted B-lymphocyte maturation into plasmatic cells.     

Atomic force microscopy fishing of GP120 on immobilized aptamers and its mass spectrometry identification

A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.     

Effect of myelopeptides on the synthesis of DNA and total protein in the cells of the lymphoid organs in the mouse

Using double 3H-thymidine/14C-amino acid label, the influence of myelopeptides (MP) on chromosomal DNA and total protein (without histones) synthesis has been studied in vitro in mouse lymphoid organ cells. It has been shown that MP cause a decrease in DNA labelling and a parallel increase in protein labelling in bone marrow cells and have practically no effect on these two parameters in thymus, lymph node and spleen cells. It has been found that MP have only mitogenic effect on the above-mentioned organ cells, but in combination with 2-mercaptoethanol. The data obtained show that MP possess the properties of cell-differentiating factors.     

Effects of thymosin and B-activin on lateralization of sensomotor control in rats

Characteristics of regulatory effects of B-activin and thymosin (fraction 5) on the activity of right and left hemispheres were studied in rats. The results obtained were estimated basing on the analysis of the following components of food-searching behavior: lateralization of a single conditioned-reflex response; preference of right or left paw; spontaneous rotations. Opposite effects of thymosin and B-activin were found at different stages of the food-searching behavior. Thymosin activated structures of the left hemisphere. A conclusion is made on the possibility of application of these drugs for targeted influencing the balance of intra-hemispheric activity in the brain functional pathology and during the compensatory process.     

Endogenous immunoregulatory peptides (myelopeptides): structure, function, and mechanism of action

Previously unknown bioregulators from bone marrow, myelopeptides, were isolated, identified, and synthesized, and their biological properties and mechanism of action were studied in detail. Phe-Leu-Gly-Phe-Pro-Thr (MP-1) manifests an immunocorrecting effect by restoring the level of antibody production in animals suffering from immunodeficiencies of various etiologies; Leu-Val-Val-Tyr-Pro-Trp (MP-2) manifests an antitumor effect by abolishing the inhibitory action of tumor cells on the functional activity of T-lymphocytes; Leu-Val-Cys-Tyr-Pro-Gln (MP-3) stimulates phagocytosis by macrophages and, in this way, protects animals from infections caused by pathogenic microorganisms; and Phe-Pro-Arg-Ile-Met-Thr-Pro (MP-4) is a new factor of cell differentiation inducing terminal cell differentiation in the HL-60 and K-562 leukemic cell lines.     

Effect of myelopeptides on physiological and pathological pain

The action of bone marrow low-molecular peptides (myelopeptides) was studied in the models of physiologic and pathologic pain. Myelopeptides were demonstrated to have a pronounced analgetic effect: they increased the latent period of the rats' response in the hot plate test (physiologic pain) and suppressed severe spinal pain syndrome induced by the generator of pathologically enhanced excitation in the dorsal horn of the spinal cord (pathologic pain). In the experiments with naloxone (an opiate receptor blocker) the data on the opiate properties of myelopeptides were further substantiated. The analgetic effect of myelopeptides can be compared to that of morphine and promedol. Myelopeptides even in considerable doses did not have the side effects characteristic of the majority of opiate analgesics. Therefore, they may be recommended for clinical trials.     

Detection of inactive or less active forms of tyrosine hydroxylase in human adrenals by a sandwich enzyme immunoassay

A sensitive sandwich enzyme immunoassay for tyrosine hydroxylase (TH) from bovine and human adrenals has been developed. Anti-TH antibody was prepared from bovine adrenal TH. The assay system consisted of an antibody F(ab')2 immobilized on polystyrene beads as a solid phase and of beta-D-galactosidase-conjugated antibody. This method was highly sensitive and specific for the assay of TH. Human adrenal TH level was determined by similar sensitivity as bovine adrenal TH, suggesting the presence of common antigenic sites between human and bovine adrenal enzymes. The presence of inactive or less active forms of TH in human adrenals was revealed by purification of the enzyme and monitoring with this enzyme immunoassay as well as with enzyme activity assay.     

Characterization of monoclonal antidigoxin antibodies immobilized to a solid support

A high-affinity monoclonal antidigoxin antibody, produced by somatic cell fusion, was amplified by the formation of ascites. Purification from ascites was accomplished by affinity chromatography by passing the ascites over a digitoxin-amine-agarose column. Affinity-purified antidigoxin antibody was coupled to a pellicular microbead at concentrations of 10, 25, 50, and 100 mg/g bead. The immobilized antibody was characterized for binding affinity, for specificity to other cardiac glycosides, and for binding capacity. There were no changes in the binding affinity observed for the immobilized antibody when compared to that of the antibody grown in culture media. Binding capacities for the immobilized antibody were decreased from calculated theoretical values. Saturating the microbead with increasing concentrations of antibody lowered the binding efficiency of the antibody from 32 to 22% of theoretical values. Attempts to improve the binding capacity by immobilizing antibodies to the microbead at the immunoglobulin carbohydrate by periodate oxidation were unsuccessful. These data demonstrate that antidrug antibodies immobilized on solid supports remain functional and may have the capability of removing drug from biological fluids passed over the support.     

Covert cross reactants in a two-site immunoassay studied with monoclonal antibodies

A one-step two-site immunoradiometric assay for the measurement of free β subunit of human chorionic gonadotrophin (β-hCG) was developed using monoclonal antibodies. The immobilized antibody was specific for free β subunit and the radiolabeled antibody recognized both intact human chorionic gonadotrophin (hCG) and free β subunit. Although the level of hCG “cross-reaction” was low when studied using conventional techniques, the apparent β-hCG content of samples was found to be inversely proportional to the hCG level. From both experimental evidence and computer simulation studies this was found to be due to the binding of hCG to the limited amount of ¹²⁵I-labeled antibody present. The term covert cross reactants has been introduced to describe substances which bind to only one of the antibodies in a two-site immunoassay. When establishing such an assay the effect of covert cross reactants on the response of an analyte should be investigated.     

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.     

Effects of high and low molecular soluble factors of the bone marrow on antibody formation and pain sensitivity in animals

In addition to the immunostimulating activity, bone marrow mediators, myelopeptides (MP) show the dose-dependent effect on the development of pain sensitivity in mice. When injected in nanogram amounts, MP induce hyperalgesia and 3-9 fold higher production of antibodies against SREC. When injected in milligram amounts, they exhibit hypoalgesic effect and no influence on antibody production. Immunostimulating effect in MP (mol, mass less than 1 KD, fraction 3) is accompanied with hypoalgesia. Bone marrow factors of mol. masses 40-150 KD (fraction 1) eluted at Sephadex G-25 gel-filtration before MP enhance the pain sensitivity tHreshold and show a potent immunodepressive effect. Thus the bone marrow factors are capable of exhibiting the opposite effects on the immune system in the pain control system that evidences the tight interrelation between these systems.