Quantification of endogenous alpha- and gamma-endorphins in rat brain by liquid chromatography-tandem mass spectrometry.

Quantification of alpha- and gamma-endorphins in rat brain using liquid chromatography-electrospray ionization-tandem mass spectrometry is described. [D-Ala(2)]-gamma-endorphin is used as an internal standard. The precursor-to-product ion MRM transitions for alpha-endorphin, gamma-endorphin, and [D-Ala(2)]-gamma-endorphin were m/z 873.6-->429.6; 929.6-->542.3; 936.6-->542.3, respectively. The method was validated in terms of linearity, specificity, sensitivity, recovery, precision, and accuracy. The assay was linear over a concentration range of 0.1-200 ng/mL with the limit-of-detection of 0.03 ng/mL and limit-of-quantification of 0.1 ng/mL. The endogenous concentrations of alpha- and gamma-endorphins in rat brains were 13.8+/-0.57 (mean+/-SD; n=5) and 2.5+/-0.43 ng/g of wet tissue weight, respectively.

Accurate mass measurement in nano-electrospray ionization mass spectrometry by alternate switching of high voltage between sample and reference sprayers.

An electrospray dual sprayer, which generates separate sample and reference sprays by alternately switching the high voltage between the two sprayers, is described. The technique permits accurate mass measurements in nano-electrospray ionization mass spectrometry (ESI-MS) to be obtained using a quadrupole/orthogonal acceleration time-of-flight mass spectrometer (Q-TOF). Similar to the method employed with a dual ESI source (Wolff JC et al., Anal. Chem. 2001; 73: 2605), the two sprays are orthogonal with respect to each other, but can be independently sampled without any baffle between these sprays. The reference sprayer is used in the original configuration of the ESI source and was optimized for a 1-2 muL/min flow, whereas the sample sprayer can be either a conventional glass capillary or a borosilicate tip of the type used for nano-ESI. Both sprayers can be positioned close to the cone so as to give maximum ion currents. The sample and reference sprays are independently generated by raising the potentials on the sample and reference sprayers to 1.4 and 3.0 kV, respectively; the high voltages can be rapidly turned on and off in ca. 1 ms. A nano-ESI-MS or nano-flow LC/ESI-MS experiment using a Q-TOF coupled with the above system gave mass accuracies within 3 ppm for measurements of ions up to m/z 1000 using subpicomole samples.

Specific cleavage of amino side chains of serine and threonine in peptides and proteins with S-ethyltrifluorothioacetate vapor.

A vapor of S-ethyltrifluorothioacetate was found to specifically cleave the amino side of serine and threonine peptide bonds. The cleavage reactions were carried out at 50 degrees C for 6 h-24 h or at 30 degrees C for 24 h. When vapors were generated in a solution containing several conventional organic solvents, the cleavage reactions were reduced or stopped, or modification took place. When the reagent vapor was made in an aqueous solution, the cleavage reaction at glycine residues was enhanced. This reagent did not oxidize any amino acid residues, such as methionine, cysteine and tryptophan. The cleavage was also effective on proteins on membranes blotted or electroblotted from polyacrylamide gels. This method therefore may be used for the peptide mass fingerprinting [Patterson, S. D. (1995) Electrophoresis 16, 1104-1114] after two-dimensional electrophoresis.

beta-Endorphin enhances polyamine transport in human lymphocytes.

Opioid peptides, such as beta-endorphin (beta-end), are capable of modulating in vitro proliferative response of lymphocytes. We attempted to determine the role of extracellular polyamines in the regulation of immune responses to opioid peptides by measuring the extent of polyamine uptake as adaptional response to cell activation. beta-end dose-dependently enhanced the incorporation of radioactive spermidine and spermine. When the cells were depleted of spermidine, with addition of specific inhibitors of both biosynthesis and interconversion pathway, a large increase in the incorporation of radioactive spermidine was observed. This effect appeares to be specific for beta-end, although a non-opiate-specific receptor could be involved, since beta-end-enhanced incorporation of radioactive spermidine is not blocked by naloxone. We conclude that the enhancement of polyamine incorporation may be considered as an integral component of lymphocyte activation by beta-end.

Study of antigen-binding properties of bispecific antibodies.

Antigen-binding properties of bispecfic antibodies (bAbs) produced by mouse hybrid hybridomas were studied. One of the bAbs held binding sites for two different antigens with relatively high molecular mass: human IgG (M(r) approximately 160,000) and horseradish peroxidase (HRP, M(r) approximately 40,000). Another bAbs showed specificity to antigens differing in molecular mass by more than an order of magnitude: peptide alpha-endorphin (END, M(r) approximately 1600) and HRP (M(r) approximately 40,000). The studied antibodies also contained different immunoglobulin chains. Both heavy chains of the anti-IgG/HRP bAbs molecule were of mouse subclass IgG1. Anti-END/HRP bAbs was formed by a combination of heavy chains which belong to two subclass of IgG: IgG2a and IgG1. bAbs were purified from ascitic fluid by a two-step affinity chromatography on columns with Sepharose-4B conjugated with the corresponding antigen. Radioimmune and immunoenzyme assays were used to analyze antigen-antibody binding and equilibrium constants of association (Ka) for each parental antibody and bAbs were determined by the Scatchard method. No significant changes in the affinity of bAbs antigen-binding sites were observed as compared to the corresponding parental antibodies. It was also shown that bAbs interaction with an excess of one of the antigens did not affect binding of the other antigen to the second bAbs site. Two-dimensional gel electrophoresis was used to analyze the composition of bAbs light and heavy chains specific to END/HRP. This analysis corroborated that bAbs molecules contained light and heavy chains from both parental hybridomas. Hence, it was demonstrated that the hybridoma fusion method can provide bispecific IgG molecules fully preserving antigen binding properties of the parental antibodies.

Characterization of unstable intermediates and oxidized products formed during cyanogen bromide cleavage of peptides and proteins by electrospray mass spectrometry.

Products formed during cyanogen bromide (CNBr) digestion of alpha-endorphin, beta-endorphin, and horse heart myoglobin are examined using reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. It is demonstrated that unstable intermediate reaction products may be formed, as well as oxidized products when the CNBr reaction is performed in 0.1% TFA in water/acetonitrile (6:4 v/v) and that, under other conditions commonly employed for the CNBr cleavage reaction, unstable intermediate products are also generated. The formation of the expected cleavage products is found to be improved by adjusting the hydrolysis conditions. The structure of the intermediate formed from alpha-endorphin is examined using electrospray mass spectrometry in combination with low-energy collision-induced dissociation and tandem mass spectrometry and is shown to have a cyclic hydrated homoserine iminolactone part. The results obtained in this study explain the formation of partially cleaved proteins in the case of Met-Thr-containing sequences, which likely have a cyclic hydrated homoserine iminolactone part instead of the putative homoserine residue.

Effects of substance P and selected other neuropeptides on the synthesis of interleukin-1 beta and interleukin-6 in human monocytes: a re-examination.

Recent studies have suggested that substance P (SP) and some other neuropeptides are able to induce the synthesis of the proinflammatory cytokines interleukin-1 (IL-1) and interleukin-6 (IL-6) in peripheral blood mononuclear cells. In the present study, we re-examined these findings by using a completely endotoxin-free monocyte cultivation system. We demonstrate that the neuropeptides SP, vasoactive intestinal peptide, substance K. cholecytokinine, alpha-endorphin and beta-endorphin are consistently unable to induce the synthesis of IL-1 and IL-6 in human peripheral blood monocytes. However, low amounts of LPS (1 pg/ml) synergized with SP to induce IL-6 mRNA expression. In contrast to its lack of effect in monocytes, we were able to confirm the ability of SP to induce cytokine synthesis in astrocytic cells. Our results raise questions about previous results claiming a neuropeptide-induced synthesis of proinflammatory cytokines in human monocytes. In conjunction with other studies, we suggest that undetected levels of endotoxin/LPS in the culture medium may have been primarily responsible for results suggesting an inductive effect of neuropeptides on cytokine synthesis in monocytes.

Neuropeptides accentuate interleukin-4 induced human immunoglobuline E synthesis in vitro.

Corticotropin releasing factor, adrenocorticotropic hormone (ACTH) and alpha-melanocyte stimulating hormone either inhibit or enhance in a dose-dependent fashion an interleukin-4 (IL-4) driven human IgE synthesis in vitro. Here, we show that culture conditions strongly influence the earlier observed dose- and donor-dependent effects of adrenocorticotropic hormone. The effect of ACTH on IgE synthesis became only apparent late during culture periods, suggesting an indirect effect via the cellular microenvironment rather than by acting directly at the level of B-cell isotype switching. Thus, we studied other proopiomelanocortin (POMC) derived peptides and neuropeptides known to influence the cellular microenvironment. Indeed, similar modulatory effects on IgE synthesis were also observed by the addition of other proopiomelanocortin-derived peptides such as alpha-, beta-, and gamma-endorphins as well as by the opioid binding pentapeptide Leu-enkephalin. Furthermore the neuropeptide substance P accentuated an IL-4 or an IL-4 and anti-CD40 antibody driven class switch to IgE. In contrast to ACTH, substance P interfered not only with IgE synthesis but also with the synthesis of the other immunoglobulin isotypes. Thus, systemically acting neuroendocrine peptides such as ACTH and locally acting neuropeptides such as the enkephalins and substance P can modulate the magnitude of an IL-4 induced IgE response.

[Bispecific monoclonal antibodies: the isolation and study of their antigen-binding properties]. / Monoklonal'nye bispetsificheskie antitela: poluchenie i izuchenie antigensviazyvaiushchikh svoistv.

Bifunctional antibodies (bABs) having a double specificity to alpha-endorphin (alpha-END) and horseradish peroxidase (HRP) were produced by hybridoma technology. The antibodies constituted about 28-29% of all immunologically active IgG secreted by hybrid hybridoma (quadroma). The quadroma was isolated by fusion of two murine hybridomas (anti-HRP and anti-alpha-END) with distinct phenotypes: double mutant AMD(R)/NAT(S) and its wild type. To produce the double mutant phenotype, an actinomycin D-resistant (AMD(R)) mouse myeloma was used to initiate one of the parental hybridomas. bABs were purified from quadroma culture medium and ascitic fluids by sequential HRP-sepharose and alpha-END-sepharose affinity chromatography. With radioimmunoassay, the affinity of the individual anti-alpha-END combining sites of bABs was shown to be identical to that of parental monoclonal antibodies. Binding to the second antigen (HRP) did not affect the binding of bABs to alpha-END. bABs proved to be efficient for the determination of endorphins and their precursor proopiomelanocortin in immunohistology and immunoblotting.

Enhancement of murine bone marrow macrophage differentiation by beta-endorphin.

The present study was performed to investigate the effect of beta-endorphin on macrophage colony-stimulating factor (M-CSF)-induced differentiation of macrophages from bone marrow cells in a semisolid culture system. beta-endorphin increased the number of macrophage colonies when bone marrow cells were cultured in the presence of M-CSF plus lipopolysaccharide (LPS). This was not the case with LPS-unresponsive C3H/HeJ mouse bone marrow cells. alpha-endorphin and gamma-endorphin were as effective as beta-endorphin in enhancing the colony formation. Exogenous interleukin-1 (IL-1), but neither IL-6 nor tumor necrosis factor (TNF), collaborated with beta-endorphin even in the absence of LPS, suggesting that IL-1 is a primary mediator of the effect of LPS. Indeed, anti-IL-1 antibody abolished the collaborative effect of beta-endorphin with LPS. Moreover, IL-1 was effective even for C3H/HeJ mouse bone marrow cells. Naloxone, an antagonist of endorphins for opioid-receptors, completely abrogated the effect of beta-endorphin. In a single-cell culture system, the collaboration between beta-endorphin and IL-1 was revealed by the increase in number and size of macrophage colonies, but collaboration between beta-endorphin and LPS did not occur. These results indicate that, in mixed cell culture, beta-endorphin acts in concert with paracrinal IL-1 induced by LPS to enhance M-CSF-dependent macrophage differentiation from immature precursor cells.

Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.

High-efficiency synthesis of human alpha-endorphin and magainin in the erythrocytes of transgenic mice: a production system for therapeutic peptides.

Chemical synthesis of peptides, though feasible, is hindered by considerations of cost, purity, and efficiency of synthesizing longer chains. Here we describe a transgenic system for producing peptides of therapeutic interest as fusion proteins at low cost and high purity. Transgenic hemoglobin expression technology using the locus control region was employed to produce fusion hemoglobins in the erythrocytes of mice. The fusion hemoglobin contains the desired peptides as an extension at the C end of human alpha-globin. A protein cleavage site is inserted between the C end of the alpha-globin chain and the N-terminal residue of the desired peptide. The peptide is recovered after cleavage of the fusion protein with enzymes that recognize this cleavage signal as their substrate. Due to the selective compartmentalization of hemoglobin in the erythrocytes, purification of the fusion hemoglobin is easy and efficient. Because of its compact and highly ordered structure, the internal sites of hemoglobin are resistant to protease digestion and the desired peptide is efficiently released and recovered. The applicability of this approach was established by producing a 16-mer alpha-endorphin peptide and a 26-mer magainin peptide in transgenic mice. Transgenic animals and their progeny expressing these fusion proteins remain health, even when the fusion protein is expressed at > 25% of the total hemoglobin in the erythrocytes. Additional applications and potential improvements of this methodology are discussed.

Identification of distinct sites of beta-endorphin that stimulate lymphokine production by murine CD4+ T cells.

We recently demonstrated that the opioid peptide beta-endorphin (beta-End) has the capacity to stimulate interleukin-2 (IL-2) and IL-4 production by murine CD4+ T cells. Since opioid peptides have been demonstrated to contain stimulatory as well as inhibitory sites, we studied peptide fragments of beta-End to identify a moiety with exclusive stimulatory capacity. To this end, the effects of various opioid peptides on the production of IL-2, IL-4, IL-6, and interferon-gamma (IFN-gamma) by CD4+ T cells were determined. It appeared that two peptide fragments of beta-End, i.e., beta-End6-31 and beta-End18-31, that lack the N-terminal enkephalin part, enhanced IL-2 and IL-4 production to a similar extent as intact beta-End, indicating that the N-terminal part is not involved in the stimulating effects of beta-End. Also the production of IL-6 and IFN-gamma was increased by these peptides. By contrast, the fragments beta-End24-31 and beta-End28-31 did not stimulate the production of the cytokines. Surprisingly, also alpha-End, which is equivalent to beta-End1-16 and hence lacks the sequence comprising amino acids 18 through 31, was stimulatory. This effect was not prevented by naloxone, indicating that opioid receptors were not involved. Moreover, methionine-enkephalin (Met-Enk), which binds to opioid receptors, did not affect cytokine production. Because both alpha-End and beta-End18-31 stimulate cytokine production by CD4+ T cells and do not overlap is sequence, it is concluded that at least two distinct sites of beta-End can exert stimulating effects on cytokine production.

Role of opioid peptides in the regulation of cytokine production by murine CD4+ T cells.

The presence of the opioid peptides alpha- and beta-endorphin (-End) but not methionine enkephalin (Met-enk) in in vitro cultures of purified CD4+ T cells, stimulated with concanavalin A in the presence of irradiated spleen cells, resulted in a threefold stimulation of IL-2, IL-4, and IFN-gamma production. The stimulating effect was dependent on the concentration of the peptides and reached optimal values in the dose range from 10(-12) to 10(-10) M. Similar results were obtained when purified CD4+ T cells were stimulated with immobilized anti-CD3, indicating a direct effect of opioid peptides on CD4+ T cells. Moreover, in this system a twofold enhancement of IL-6, but not IL-1, secretion was observed. These stimulatory effects were not mediated through opioid receptors since the peptide fragment beta-End6-31 that lacks the N-terminal opioid receptor binding part was still stimulatory. This is in agreement with our finding that beta-End did not affect cAMP, as described for the triggering of classical opioid receptors. Experiments undertaken to reveal the mechanism of action of opioid peptides suggest an overall enhancement of lymphokine production: (1) enhancement of IL-4 production occurred also in the presence of excess IL-2; and (2) neither IL-1 receptor-antagonizing protein nor anti-IL-6 were capable to abrogate the stimulatory effect on IL-2 and IL-4 production. Finally, the presence and activity of opioid receptors in cultures of CD4+ T cells were substantiated by the fact that the opioid receptor antagonist naloxone by itself enhanced cytokine synthesis, which points to the endogenous production by lymphocytes of down-regulating opioid peptides.

[The interaction of bifunctional monoclonal antibodies with antigens studied by a radioimmunological method]. / Izuchenie vzaimodeistviia bifunktsional'nykh monoklonal'nykh antitel s antigenami radioimmunologicheskim metodom.

The quadroma produced bifunctional antibodies (bAbs) with double specificity to alpha-endorphin (alpha-END) and horseradish peroxidase (HRP) were compared with the parental anti-alpha-END monoclonal antibodies (mAbs) in respect to their binding to alpha-END. bAbs were purified from quadroma culture medium by sequential HRP-sepharose and alpha-END-sepharose affinity chromatography. Using radioimmunological method the affinity of the individual anti-alpha-END combining sites of bAbs was shown to be identical to that of parental mAbs. Binding to the second antigen (HRP) didn't affect binding of bAbs to alpha-END.

Construction of a quadroma to alpha-endorphin/horseradish peroxidase using an actinomycin D-resistant mouse myeloma cell line.

A hybrid hybridoma (quadroma), secreting antibodies with double specificity to alpha-endorphin (alpha-EP) and horseradish peroxidase (HRP), has been produced. The bispecific antibodies constituted about 28-29% of all immunologically active IgG, produced by quadroma. The quadroma was isolated by fusion of two mouse hybridomas (anti-HRP and anti-alpha-EP) with distinct phenotypes: double mutant AMDR/HAT(S), and wild type (AMDS/HATR). A novel strategy for the construction of a double-mutant was applied, based on the use of an actinomycin D-resistant (AMDR) mouse myeloma for initiation of one of the parental hybridomas.

Endorphin-like immunoreactivities in uncultured and cultured human peripheral blood mononuclear cells.

It has been reported that cells of the immune system produce and release considerable amounts of pro-opiomelanocortin (POMC) -derived peptides in response to coculture with a variety of stimulatory agents. The present study investigated whether extracts of human peripheral blood mononuclear cells (PBMC) contain immunoreactivity for beta-endorphin (beta E) and related peptides. Using four endorphin RIA systems with different specificities, extracts of freshly isolated PBMC and PBMC cultured in the presence or absence of mitogens or of corticotropin releasing factor (CRF) and vasopressin (VP), were analyzed. With a radioimmunoassay (RIA) system directed to the midportion of beta E, immunoreactivity (MP beta E-IR) was readily detectable, although the concentration was extremely low (ca. 200 pg/10(7) cells). beta E immunoreactivity (beta E-IR) and alpha-endorphin immunoreactivity (alpha E-IR), as determined in C-terminally directed RIA systems, were present in even lower concentrations. gamma-Endorphin immunoreactivity (gamma E-IR) was hardly detectable. Of subsets enriched in T-cells, B-cells or monocytes, the highest concentration of MP beta E-IR was detected in extracts of monocytes. Coculture of PBMC with the mitogen Concanavalin A (Con A) or Phytohaemagglutinin (PHA) increased the amount of MP beta E-IR in extracts of the cells. No increase in alpha E-IR, however, was detected, whereas beta E-IR was only increased in extracts of cells cultured in the presence of Con A. No increase, in any of the immunoreactivities, was observed in extracts of PBMC cultured with bacterial lipopolysaccharide (LPS) or with the combination of CRF and VP, both stimuli that have been reported to induce POMC peptides in cultured PBMC. The present data show that human PBMC contain endorphin-like immunoreactivity, but in very small amounts. The extremely low concentrations and the ineffectiveness of LPS and the combination of CRF and VP to increase the endorphin-like immunoreactivity raise questions about the reported capacity of PBMC to synthesize POMC-derived peptides.

[A new approach to the design of hybrid hybridomas based on the use of an actinomycin D-resistant line of murine myeloma]. / Novyi podkhod k konstruirovaniiu gibridnykh gibridom, osnovannyi na ispol'zovanii aktinomitsin-D-rezistentnoi linii mielomy myshi.

The hybrid hybridomas (tetradomas) were produced from the fusion of the double mutant actinomycin Dr (ADr)/HATs hybridoma to horseradish peroxidase (HRP) and wild type hybridoma to alpha-endorphin (EP). The double mutant phenotype was constructed using the new strategy, based on the fusion of immune mouse splenocytes with mouse myeloma (X63.Ag8, 653) cell variants, made resistant to 30 ng/ml of AD by stepwise selection. This allowed the direct introduction of the dominant selective marker (ADr) into the hybrid cells. Tetradomas secreted the bispecific monoclonal antibodies (bi Mabs), simultaneously binding to EP and HRP in double antigen ELISA, the ELISA plates covered with EP-bovine serum albumin conjugate. Using rat pituitary the bi Mabs were shown to be effective for immunostaining of EP-producing cells. EP-producing cells.

Endogenous opioid peptides and blood pressure regulation during controlled, stepwise hemorrhagic hypotension.

In the present study, the role of the endogenous opioid peptide systems in the regulation of blood pressure during standardized, stepwise hemorrhagic hypotension was investigated in anesthetized rats. Central as well as peripheral administration of naloxone resulted in an increase in the bleeding volumes required to reduce blood pressure. Bleeding volumes also increased after the peripheral injection of naloxone methobromide, an analog of naloxone that does not readily cross the blood-brain barrier. Following central administration of antisera against beta- and alpha-endorphin and dynorphin A(1-13), the amount of blood that had to be withdrawn to induce hypotension was elevated. In rats treated with an antiserum against [Met5] enkephalin or gamma-endorphin, bleeding volumes did not differ from those of rats treated with control serum. These data indicate that activation of central and possibly also of peripheral opiate receptors plays a role in the control of blood pressure during blood loss. Dynorphin A(1-13), beta- and alpha-endorphin, or closely related peptides might be the endogenous ligands for the receptors that are blocked by naloxone.