Molybdic and tungstic heteropolyanions for "ionic fixation" of acetylcholine in cholinergic motor nerve terminals

The treatment of neuromuscular junctions with phosphomolybdic acid (PMA) and silicotungstic acid (STA) heteropolyanions permits the visualization of electron dense precipitates in the synaptic vesicles of the cholinergic motor nerve terminals. At the light microscopic level, the uncolored molybdenum salt is visualized after reduction to molybdenum blue. The blue coloration is confined to the nerve terminals. Since PMA and STA are known as strong precipitating agents of quaternary ammonium compounds (cations) it is supposed that they have insolubilized in situ the acetylcholine (Ach) of the synaptic vesicles by means of a rapid ionic interaction. Furthermore, in spite of the strong acidity of PMA and STA solutions, the ultrastructure of the treated tissue is not significantly altered but on the contrary seems to be well preserved. The ionic insolubilization of Ach, added to the good preservation of the ultrastructure prompted us to use the term "ionic fixation".     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

In vivo and in vitro synergistic antitumor effect of interleukin-2-cultured tumor-bearer spleen cells and immune fresh spleen cells

Summary The synergistic antitumor effect of interleukin-2(IL-2)-cultured tumor-bearer spleen cells (cultured lymphocytes) and immune fresh spleen cells was examined. Tumor-bearer cultured lymphocytes were obtained by culturing BALB/c spleen cells from syngeneic MOPC104E-tumor-bearing mice for 11 days with crude IL-2 and a soluble tumor extract. These cultured lymphocytes had weak antitumor activity when transferred i.p. into tumor-bearing mice that had been inoculated i.p. with 10⁵ tumor cells 5 days previously. Immune fresh spleen cells, obtained from mice in complete remission after the treatment with cyclophosphamide, also had weak antitumor activity when transferred at the same schedule. The cultured cells and the fresh cells, mixed together before transfer, significantly augmented the therapeutic effect. At least 1×10⁷ tumor-bearer cultured lymphocytes and 4×10⁷ immune cells were needed for the synergistic effect. A tumor-specific combination was needed for both cultured and fresh cells. The effective subpopulation of tumor-bearer cultured lymphocytes was a cytotoxic one from an Lyt2⁺ precursor, and that of the immune fresh spleen cells was noncytotoxic, Lytl⁺ and Lyt2⁺ T-cells.A similar synergistic effect was also observed during in vitro coculture of tumor-bearer and immune cells. Cytotoxicity, as assessed by the ⁵¹Cr-release test, of tumor-bearer IL-2-cultured lymphocytes was maintained most effectively after 3 or 4 days of culture without IL-2 when the lymphocytes were cocultured with immune fresh spleen cells and tumor cells.     

Lectin histochemistry in rat thyroid tumours

The thyroid tumours and background goiterous and adenomatous lesions induced in rats with diisopropanolnitrosamine (DIPN) plus methylthiouracil (MTU), and regenerative thyroid tissues after wounding were studied by lectin histochemistry. Ten weeks after cessation of the carcinogen treatments, carcinomas invading the surrounding tissues and blood vessels (13/20) and papillary micronodules (11/20) were formed in the thyroid tissues. In general, the carcinoma lesion was solitary, and the papillary micronodules were multiple in a single thyroid gland. Among the lectins tested, Maclura pomifera (MPA) and Solanum tuberosum (STA) showed specific binding with both carcinoma and papillary micronodule lesions, but not with the background goiterous and adenomatous lesions and regenerative thyroid tissues. The former both lesions showed higher labelling indices with BUdR or 3H-thymidine and poorer thyroglobulin accumulation than the latters, thereby indicating their enhanced proliferative capability and depressed potency of cyto-differentiation. The common cytological and histochemical properties of carcinoma lesions and papillary micronodules allow us to regard the latter as pre-invading carcinoma lesions. The lectins MPA and STA may be, therefore, used as the specific markers of malignancy in rat thyroid carcinogenesis.     

Immunochemical evidence that myosin I heavy chain-like protein is identical to the 110-kilodalton brush-border protein

In a previous study, we identified a new mammalian myosin heavy chain, termed myosin I heavy chain-like protein (MIHC), by molecular cloning of a bovine intestinal cDNA clone. In this investigation, we examined the relationship between MIHC and the 110-kDa intestinal brush-border protein, which possesses a myosin-like ATPase activity. We raised antibodies against a chemically synthesized oligopeptide representing a part of the MIHC sequence. These antibodies reacted specifically in immunoblots with the 110-kDa protein in both purified 110-kDa protein-calmodulin complex and crude microvillar protein extracts. Staining of tissue sections with these antibodies was specifically localized to the brush-border microvilli of small intestines, indicating an identical cellular localization for both MIHC and the 110-kDa protein. Furthermore, analysis of the MIHC sequence revealed two putative calmodulin-binding sites, which is consistent with the fact that the 110-kDa protein forms a complex with calmodulin. These results strongly support the conclusion that MIHC is identical to the 110-kDa protein and suggest that not only the conventional myosin system but also the MIHC (110-kDa protein)-calmodulin complex may play an important role in ATP-dependent and Ca2+-induced brush-border contraction.     

Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme

Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.