The heterocytotoxicity of human serum. I. Activation of the alternative complement pathway by heterologous target cells.

Human serum induces cytolysis of mouse thymus and thymoma cells, and cytostasis of mouse bone marrow and spleen cells, and various methylcholanthrene-induced tumour cells. The latter was manifested by deficient metabolic activity when cultured in the presence of fresh human sera. Decomplementation procedures demonstrated that these heterocytotoxic effects are mediated in part via activation of the alternative complement pathway in human serum samples. The presence of properdin and C3 on the target cell surface was confirmed by immune adherence and indirect immunofluorescent tests. Activation of the alternative complement pathway was elicited by incubation of the human serum with the relevant target cells, resulting in the appearance of the cathodal migrating fragment of the factor B, denoting complement activation. The following publication will present evidence that activation of the alternative complement pathway takes place via an antibody-independent mechanism acting at the cell surface. These and other observations in the literature raise the possibility that activation of the alternative complement pathway by surface cell receptors on tumour cells represents a mechanism of natural immunity versus tumours.     

Metal ion-biomolecule interactions. III. Versatility of metal ion binding to imidazoles. Synthesis and 1H nmr spectroscopic properties of N- and C-bound mercury(II) complexes

Methylmercury(II) and mercury(II) complexes of imidazole (1), 1-methylimidazole (2), and the 1,3-dimethylimidazolium ion (3) have been prepared in aqueous or ethanolic solution. Elemental analysis and ¹H nmr spectroscopy have been used to characterize the complexes. The MeHg (Me = methyl) binding sites have been identified as N₁, N₃ (1), N₃, C₂ (2), and C₂ (3). Reaction with HgO leads to the formation of Hg-bridged complexes of the type Im-Hg-Im, (Im = imidazole), where bonding occurs through N₁ (1) and C₂ (3); the latter is also formed as a result of symmetrization of the C₂-bound MeHg complex. The formation of the C₂-bound (carbene) complexes is discussed in terms of the increased acidity of the C₂ proton resulting from coordination of an electrophilic species at N₃. Based on electrostatic considerations, there appears to be a “minimum degree of activation” required before C₂ bonding can occur, which explains the lack of this coordination mode in 1. ¹⁹⁹Hg-¹H spin-spin coupling (⁴J) is observed for C-bound mercury, but not for N-bound mercury, which is interpreted in terms of a decreased ligand exchange rate in the former case, due to the greater stability of the Hg-C bond. ²J coupling constants measured in (CD₃)₂SO for a number of MeHg complexes of heterocyclic ligands (including the imidazoles of the present study) correlate well with the ligand pK_a (25°C, aqueous solution), according to ²J = ?3.88 pK_a + 248.5. Results in the present work are discussed in relation to our previous work with nucleosides. The significance of the results to biological systems is considered.     

Metal Ion-Biomolecule Interactions. VIII: Methylmercury(II) Complexes of 8-Thioguanosine. Synthesis and Spectroscopic Investigations

The reaction of 8-thioguanosine (8-thioGuo^H₂ with methylmercury(II) has been shown to give rise to 1:1 (neutral and cationic), 1:2 (neutral and cationic), and 1:3 (cationic) complexes of the type [CH₃Hg(8-thioGuoH)], [(CH₃Hg(8-thioGuoH₂)]NO₃, [(CH₃Hg)₂(8-thioGuo)], [(CH₃Hg)₂(8-thioGuoH)]NO₃ and [(CH₃Hg)₃(8-thioGuo)]NO₃, depending upon the reactant stoichiometry and pH. ¹H NMR, ¹³C NMR, and IR, as well as analytical data were used to characterize the complexes. Coupling of methylmercury(II)-protons to mercury-199 has been observed in all compounds. The magnitude of the coupling, ²J(¹H-¹⁹⁹Hg), is strongly dependent on the nature of the ligand bonded to the methylmercury(II) group and correlates with the ¹³C chemical shifts of coordinated CH₃Hg(II) at the different binding sites.     

Metal ion-biomolecule interactions. XII. 1H and 13C NMR evidence for the preferred reaction of thymidine over guanosine in exchange and competition reactions with Mercury(II) and Methylmercury(II)

Mercury(II) bridge complexes of the type [Nuc-Hg-Nuc] (Nuc = thymidine or guanosine), and methylmercury(II) complexes of thymidine and guanosine of the type [CH₃Hg(Nuc)], have been prepared under appropriate conditions of pH and reactant's stochiometry in acqueous soluton. The various complexes have been characterized by ¹H and ¹³C NMR and used as probes, in competition and exchange studies, to establish the relative affinities of Hg(II) and CH₃Hg(II) towards the nucleosides guanosine and thymidine. These studies have confirmed that Hg(II) and CH₃Hg(II) bind to N₃ of thymidine in preference to N₁ of guanosine. The studies further show that reactions of mercury(II) with the nucleosides are thermodynamically controlled; the preperential binding reflects the relative stabilities of the respective complexes.     

Regulation of humoral immune responses by a bone marrow-derived glycolipid-like molecule

Bone marrow cells have been shown to nonspecifically suppress primary in vitro antibody responses. This suppression appears to be mediated by a low-molecular-weight soluble factor, B-SF which was released from a fraction of cells of similar size to the suppressor as obtained by velocity sedimentation. Like the suppressor cell, B-SF was also shown to be effective very early in the immune response. It was produced by all strains of mice tested and functioned across strain barriers. Characterization of the active suppressor molecule showed it to be a highly heat-stable, nonsialic acid-containing glycolipid of 1000 to 35000 daltons in molecular weight. Recovery of the purified suppressor from thin-layer chromatography plates was achieved indicating that the major glycolipid component visualized on TLC is likely the active suppressor molecule. The characteristics of this suppressor may show it to be a fundamental immune regulatory mechanism.     

Galvinoxyl: a new spin probe for detecting phospholipid phase transitions.

Galvinexyl is a stable paramagnetic molecule which can be easily incorporated into phospholipid vesicles (liposomes). The csr spectrum from such labeled vesicles displays striking changes in the region of the phospholipid phase transition. These changes result from an enhancement in the tumbling motion of galvinoxyl molecules as a consequence of the “loosening” of the lipid environment that occurs at the phase transition.     

Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.     

Addendum

A number of solid complexes of theophylline with CH₃Hg¹¹, Hg¹¹, as well as both CH₃Hg¹¹ and Hg¹¹, have been isolated from aqueous solution as the result of the reactions of theophylline and mercury-containing species at the appropriate pH and mole ratios of reactants. The complexes have been characterized by elemental analysis, ¹H and ¹³C NMR and infrared spectroscopy, and X-ray crystal structure analysis. The results obtained indicate that the initial site of mercury binding is strongly pH dependent. No evidence could be found for CH₃Hg¹¹ substitution at the C₈ position in theophylline, contrasting with the situation in xanthosine and inosine.     

The purification of methionine sulfoxide reductase from Escherichia coli

A sensitive assay, based on the acylation of tRNA^Met, has been developed to measure the enzymatic reduction of methionine sulfoxide to methionine. Using this assay, methionine sulfoxide reductase has been purified to near homogeneity from extracts of Escherichia coli.     

Quantitation and interaction of glycosaminoglycans with Alcian blue in dimethyl sulfoxide solutions.

An improved method is described for the quantitation of glycosaminoglycans separatedon cellulose acetate, stained with Alcian blue, and dissolved in a dimethyl sulfoxide solution. Standard curves are shown for all eight glycosaminoglycans. It is shown that absorption at the Alcian blue orthochromatic E_max is depressed under conditions which favor formation of dye-glycosaminoglycan complexes. The interaction between Alcian blue and the eight glycosaminoglycans was studied in dimethyl sulfoxide solutions of varying composition. It was shown that the extent of complex formation depends both on the glycosaminoglycan and the composition of the dimethyl sulfoxide solution. A dimethyl sulfoxide solution which contains 0.094 m H₂SO₄ is described which maximizes dye-glycosaminoglycan dissociation and thus the absorbance. Also, an improved staining method is described which improves dye uptake by the glycosaminoglycans and consequently increases the sensitivity of glycosaminoglycan quantitation.     

Comparative analysis of indices of central dopaminergic functions in man

Various postulated indices of central dopaminergic activity - cerebrospinal fluid (CSF) dopamine (DA), dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), plasma NA, serum prolactin, serum dopamine-β-hydroxylase (DBH), and platelet monoamine oxidase (MAO) activity - were measured in 30 drug-free inpatients. The mean values and the ranges were similar to those described in the literature. Plasma NA showed significant positive correlation with age. Significant positive correlation was found between CSF DA and its metabolites DOPAC and HVA. Serum DBH activity showed a slight but significant inverse correlation with CSF DA and its two metabolites. CSF NA showed a significant positive correlation with CSF DOPAC, but only in females. Serum DBH activity had no significant correlation either with CSF or with plasma NA levels. These findings suggest that either CSF HVA or DOPAC and DA may be useful indicators of DA metabolism in humans. Serum DBH activity may be in relationship with the central dopaminergic functions.     

The denaturation of ribonuclease A by combinations of urea and salt denaturants.

When urea is added to ribonuclease A that has already been denatured by salt (CaCl₂, LiClO₄ or LiCl were used), a second co-operative transition occurs, supporting the previous demonstration that these salts cause only partial denaturation. Also we have studied the effect of the salts on the urea denaturation, and the effect of urea on the salt denaturation. At low concentrations urea makes the salt transitions occur at lower concentrations, but at higher concentrations it changes the transition so that the completely disordered protein found in urea is produced by the salt. At low concentrations the salts actually stabilize the protein against denaturation by urea, but at higher concentrations they destabilize it. The data are presented in “phase diagrams” which are found to be very useful for such three-component systems.     

Regulatory cells in human bone marrow: Suppression of an in vitro primary antibody response

Human bone marrow (BMC) contains regulatory cells that can suppress the in vitro primary PFC response of normal allogeneic spleen or tonsillar cells and autologous peripheral blood cells. Suppression is dependent upon the dose of BMC added, but is not due to cell crowding nor to excessive cytotoxicity, and requires the presence of viable, metabolically active BMC. BMC are maximally inhibitory when added during the first 24 hr of culture and do not cause an induced shift in the kinetics of the response. Thus, suppression reflects inhibition of early inductive events in the antibody response. The target of suppression is the non-T cell, with either polyclonal activator or Ag being required for maximal suppression. DNA synthesis of normal tonsillar cells is not inhibited by BMC. Characterization of the human bone marrow-suppressor cell has shown it to be radiosensitive, E-rosette negative, Fc receptor positive, and to reside in the large, weakly adherent cell population after velocity sedimentation and in the lymphocyte-depleted fraction after sucrose density gradient separation. Pretreatment of the bone marrow-suppressor cell with anti-human thymocyte serum does not abrogate suppression. We speculate on a possible physiologic role for this cell.     

Activated T lymphocytes resist the toxic effects of the adenosine deaminase inhibitor, 2-deoxycoformycin

Tonsil lymphocytes from three adults and three children were examined for immunoglobulin (Ig) production before and after Epstein-Barr virus (EBV) transformation. T-cell depletion was required to obtain cell lines from EBV-seropositive individuals. Cytoplasmic Ig was mainly IgG in adult lymphocytes before and after transformation; IgA and IgM were more prominent after than before. IgM and IgG predominated in lymphocytes of children before and after transformation; IgA was more prominent after than before. Cytoplasmic Ig of peripheral blood lymphocytes from these individuals was mainly IgM. Secreted Ig from tonsil lymphocytes was mainly IgA or IgG; after transformation IgM predominated with adult cell lines, and IgG or IgM with cell lines from children. IgE was consistently sparse in spite of ragweed and/or grass allergies of the adults.     

Biphasic Alteration of Butyrylcholinesterase (BChE) During Prostate Cancer Development

Butyrylcholinesterase (BChE) is a plasma enzyme that hydrolyzes ghrelin and bioactive esters, suggesting a role in modulating metabolism. Serum BChE is reduced in cancer patients. In prostate cancer (PC), the down-regulation is associated with disease recurrence. Nonetheless, how BChE is expressed in PC and its impact on PC remain unclear. We report here the biphasic changes of BChE expression in PC. In vitro, BChE expression was decreased in more tumorigenic PC stem-like cells (PCSLCs), DU145, and PC3 cells compared to less tumorigenic non-stem PCs and LNCaP cells. On the other hand, BChE was expressed at a higher level in LNCaP cells than immortalized but non-tumorigenic prostate epithelial BPH-1 cells. In vivo, BChE expression was up-regulated in DU145 xenografts compared to LNCaP xenografts; DU145 cell-derived lung metastases displayed comparable levels of BChE as subcutaneous tumors. Furthermore, LNCaP xenografts produced in castrated mice exhibited a significant increase of BChE expression compared to xenografts generated in intact mice. In patients, BChE expression was down-regulated in PCs (n = 340) compared to prostate tissues (n = 86). In two independent PC populations MSKCC (n = 130) and TCGA Provisional (n = 490), BChE mRNA levels were reduced from World Health Organization grade group 1 (WHOGG 1) PCs to WHOGG 3 PCs, followed by a significant increase in WHOGG 5 PCs. The up-regulation was associated with a reduction in disease-free survival (P = .008). Collectively, we demonstrated for the first time a biphasic alteration of BChE, its down-regulation at early stage of PC and its up-regulation at advanced PC.     

Differences in proteins secreted by human fibroblasts and muscle cells in culture

Cell cultures of human skin fibroblasts, myoblasts, and fused muscle cells were grown in the presence of [¹⁴C]leucine or a mixture of [¹⁴C]amino acids. The proteins synthesised and secreted or leaked into the culture medium during radio-labelling were separated by one and two-dimensional PAGE and detected by fluorography. Four major bands of M_r 54 kD, 52 kD, 51 kD, and 49 kD were present at greatly increased concentration in fibroblast media. These fibroblast-specific polypeptides can be readily detected in myoblast/fibroblast cocultures with fibroblast content as low as 5%.     

Polysaccharides with sulfate groups are human T-cell mitogens and murine polyclonal B-cell activators (PBAs). I. Fucoidan and heparin

We reported previously that dextran sulfate and carrageenan (kappa, lambda, and iota), which are sulfated polysaccharides, were human T-cell mitogens and mouse polyclonal B-cell activators (PBA). To clarify our working hypothesis further, we used fucoidan and heparin, both sulfated polysaccharides. The following results were obtained: (1) fucoidan is human T-cell mitogen and a mouse PBA; (2) heparin is also a human T-cell mitogen and a mouse PBA, but the degree of the responses by heparin is lower than that by fucoidan; (3) helper T-cell-dependent B-cell differentiation was not observed, since both fucoidan and heparin activate OKT4⁺ cells and OKT8⁺ cells nonspecifically and suppressor T cells (OKT8⁺ cells) may inhibit the helper function of B-cell differentiation by helper T cells (OKT4⁺ cells); and (4) our working hypothesis that polysaccharides with sulfate groups are human T-cell mitogens and mouse PBAs was further strengthened. The relationship between molecular weight and sulfate groups of the polysaccharides is discussed in detail.     

Detection of lipopolysaccharides in polyacrylamide gels by transfer to nitrocellulose followed by immunoautoradiography with antibody and 125I-protein A: "LPS blotting"

Lipopolysaccharides (LPS), which constitute the somatic (O) antigen of gram-negative bacteria, were used to demonstrate the procedure of LPS blotting involving the electrophoretic transfer of electrophoretically resolved LPS from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose filters. Immobilized LPS could then be immunoautoradiographically visualized in situ by reaction with specific anti-LPS antibody and subsequent binding of radioiodinated Staphylococcus protein A. LPS blotting is expected to provide an efficient and specific means of investigating the LPS (O) antigens of gram-negative bacteria.