S-100 Protein in Clonal Astroglioma Cells Is Released by Adrenocorticotropic Hormone and Corticotropin-Like Intermediate-Lobe Peptide

S-100 protein in clonal GA-1 and C6 rat glioma cell lines was released in serum-free medium supplemented with adrenocorticotropic hormone (ACTH). The induction of S-100 protein release by ACTH was dose-dependent, showing a half-maximal release at about 5 microM, and the S-100 protein concentration in the medium increased sharply within 3 min, but slightly during further incubation. The S-100 protein release was apparently accompanied by a decrease in the membrane-bound form of S-100 protein in the cell. The S-100 protein release was induced not by the ACTH1-24 fragment, which exhibits the known effects of ACTH, but by the ACTH18-39 fragment, which is designated as corticotropin-like intermediate-lobe peptide (CLIP). These results indicate that the C-terminal half of ACTH is responsible for the S-100 protein release. The enhancement of S-100 protein release by ACTH was also observed in normal rat glioblasts. The release induced by ACTH was apparently specific to S-100 protein, because little release of the cytoplasmic enzymes, creatine kinase, and enolase was observed under the same conditions. High concentrations (5 mM) of dibutyryl cyclic AMP or dibutyryl cyclic GMP were also found to induce S-100 protein release; however, catecholamines (epinephrine, norepinephrine, isoproterenol, and dopamine), acetylcholine, and glutamic acid did not enhance the release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of plasma retinolbinding protein and prealbumin in human pancreatic islets

Summary This study was undertaken, employing the immunoenzyme method, to confirm the presence of retinol-binding protein in human pancreatic islets, and to compare its distribution with that of prealbumin, insulin, glucagon, somatostatin and pancreatic polypeptide. It was found that most islet cells contained retinol-binding protein, although centrally located cells showed stronger reactivity than those in the peripheral region. The distribution of each of the five polypeptides differed from that of retinolbinding protein, indicating that these peptides did not cross-react with anti-retinol-binding protein antibody. Islet cells which contained prealbumin, on the other hand, were mostly classified as A cells. Further studies are necessary to confirm whether the islet cells produce retinol-binding protein or only store it.     

Aminoacyl-tRNA exclusively in the 3'-isomeric form is bound to polypeptide chain elongation factor Tu

2' and 3'-O-(N-acetyl-L-phenylalanyl)adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC). From the two isomers of [3H]Phe-tRNA in equilibrium, Ac-[3H]Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was found to be 0.20:0.80. Further, [3H]Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GMP-P(NH)P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu.GTP and 0.04:0.96 in the complex with EF-Tu.GMP-P(NH)P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex.     

The rate assay of alpha-toxin assembly in membrane

Abstract A rapid and easy method to determine the 'rate' of the assembly of α-toxin from Staphylococus aureus in erythrocyte membrane was described. Upon addition of a small amount of α-toxins into erythrocyte suspension, absorbance at 700 nm decreased linearly after a short period of lag time. From the linear portion of the record the rate of the assembly of α-toxin was calculated. An optimum temperature and an optimum pH for the assembly of the toxin on erythrocyte membranes were found to be 25–30°C and pH 5.     

Guest-induced conformational change of β-cyclodextrin capped with an environmentally sensitive chromophore

A capped cyclodextrin, 6-deoxy-6-(p-hydroxy-m-nitrophenacylthio)-β-cyclodextrin, was prepared in order to detect any conformational change of the host upon the guest binding. The association constant between the cyclodextrin and 1-adamantanecarboxylate, cyclohexanecarboxylate, p-methylbenzoate, 3,3-dimethylbutyrate, or 2,2-dimethylpropionate was enhanced 20, 5.3, 3.7, 2.3, or 2.0 times, respectively, by chromophore capping. The changes in the electronic, NMR, and circular dichroism spectra as well as pK_a of this cyclodextrin upon binding of the guest strongly indicate a conformational change around the chromophoric moiety of the cyclodextrin.     

Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification

Eight murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin D (SED) were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED only or a combination of SED and either enterotoxin A (SEA) or enterotoxin E (SEE). When only SED was used as an immunogen, six MAbs were specific for SED only, whereas one MAb was reactive with both SED and SEE when both SEs were used as immunogens. One MAb reacted with SEA, SED, and SEE when both SEA and SED were used as immunogens. A MAb with the highest reactivity to SED was used to prepare an immunosorbent for purification of SED by immunoaffinity chromatography. Approximately 70% of the partially purified SED was recovered in the eluate. The purified SED was electrophoretically and antigenically pure. Immunoaffinity chromatography proved useful in the purification of SED in terms of ease of purification, percent enterotoxin, and enterotoxin purity.     

A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma

Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion-exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross-reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemically visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent M(r) of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent M(r) of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50-kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor-stimulated astroblasts, but not neuronal cells.     

Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3′:5′-monophosphate(dibutyryl cAMP) and prostaglandin E₁ (PGE₁). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE₁ (0.5 μg/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 μg/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE₁, and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine β-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.