Anti-histone antibodies in idiopathic and drug-induced lupus recognize distinct intrahistone regions

We have identified regions within core histones that are antigenic for autoantibodies in systemic lupus erythematosus (SLE) and drug-induced lupus. An immunoblotting technique was used to determine the reactivity of lupus antibodies for intact histones and for trypsin-resistant histone fragments that lack the amino- and carboxyl-terminal amino acids that are normally exposed in native nucleosomes. In SLE, the predominant anti-histone response was restricted to epitopes in the trypsin-sensitive regions. Of 20 SLE sera that had strong antibody activity for multiple intact histones, 17 showed minimal activity with any of the corresponding trypsin-resistant fragments. A markedly different pattern of reactivity was present in sera of patients with procainamide (Pr)-induced lupus in which antibodies to H2A, H2B, and the H2A-H2B complex had strong fragment activity. Interestingly, recognition of trypsin-resistant fragments was also noted in a small number of SLE sera that contained antibodies to the H2A-H2B complex. In contrast to both SLE and Pr-induced lupus, antibodies induced by hydralazine (Hy) reacted primarily with H3 and H4. Furthermore, these antibodies bound equally well to the corresponding trypsin-resistant regions that are thought to be relatively unexposed in native nucleosomes. Thus, the specificities of anti-histone antibodies in SLE, Pr-induced lupus, and Hy-induced lupus are markedly different, but in each disease reactivity appears to be restricted to a limited number of histone determinants. The data raise the possibility that autoantigen in the form of native nucleosomes may be recognized in SLE and possibly in Pr-induced lupus. In contrast, the propensity of Hy to induce autoantibodies to determinants usually not recognized in SLE or Pr-induced lupus may suggest a different immunogenic stimulus in this disease.     

Assessment of histogenesis and proliferative potential in cytologic specimens of human brain tumors. Value of immunocytochemistry and nucleolar organizer regions.

The value of immunocytochemistry and nucleolar organizer regions (NORs) for the histogenetic identification and the estimation of the proliferative potential of brain tumors was assessed by the investigation of imprint smears of 51 neurosurgical tumor specimens. A panel of five monoclonal antibodies was used to cover a broad range of immunohistochemical markers. For the assessment of NORs, a silver staining technique (AgNOR) was used. NORs were enumerated and measured by means of an interactive image analysis system. The immunocytochemical results were similar for the smears and paraffin-embedded sections for 95.6% of the investigations performed and for 76.2% of the cases. Glial fibrillary acidic protein (GFAP) was positive in 9 of 17 tumors of glial origin, but was negative in 9 metastatic tumors. Vimentin was positive in 10 of 10 and fibronectin in 9 of 10 meningiomas investigated. The number of NORs increased steadily with the increasing grade of malignancy. Especially in glioblastomas, the number of NORs per cell exhibited a wide range, which might reflect the heterogeneity of these neoplasms. Metastases revealed a higher number of NORs per cell than did glioblastomas. In the cytologic differential diagnosis of these tumors, an absence of GFAP expression combined with a high NOR count is suggestive of a metastatic tumor.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Characterization of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in human neutrophils

Phosphodiesteric cleavage of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) is required for transmembrane signaling by chemoattractants in human polymorphonuclear leukocytes (PMN). Considering the importance of PtdIns-4,5-P2 as a reservoir for second messenger substances, we have characterized the enzyme system that synthesizes this phospholipid in human PMN, consisting of kinases for phosphatidylinositol (PtdIns) and phosphatidylinositol-4-phosphate (PtdIns-4-P). The preferred phosphate donor for both enzymes was ATP as compared with GTP. The respective Km for ATP for PtdIns kinase and PtdIns-P kinase were 0.049 +/- 0.013 and 0.062 +/- 0.005 mM and for GTP were 0.242 +/- 0.016 and 0.186 +/- 0.037 mM. PtdIns stimulated the activity of PtdIns kinase to a greater extent than PtdIns-4-P kinase. PtdIns-4-P inhibited the activity of detergent-solubilized PtdIns kinase and stimulated particulate PtdIns-4-P kinase, whereas both enzymes exhibited substrate inhibition to PtdIns-4,5-P2. Mg2+ was the preferred cation for both enzymes, but the apparent Km values (4.1 +/- 0.9 mM for PtdIns kinase and 1.0 +/- 0.7 mM for PtdIns-4-P kinase) were significantly different (p less than 0.005). Mn2+ partially substituted for Mg2+, and both enzymes were inhibited by Ca2+. The polyamine spermine stimulated PtdIns-4-P kinase activity to a greater extent and at lower concentrations than PtdIns kinase. PtdIns kinase was easily solubilized in both Triton X-100 and Nonidet P-40, whereas PtdIns-4-P kinase remained in a detergent-nonextractable membrane fraction. These findings demonstrate that the enzyme system in human PMN that forms PtdIns-4,5-P2 is composed of two distinct enzymes with similar characteristics.     

The effect of a single colony of the red wood ant,Formica polyctena,on the spider fauna (Araneae) of a beech forest floor

Summary The predation on spiders in a forest ecosystem by a colony of red wood ants, Formica polyctena, was estimated using a barrier to isolate the colony. Of the ants' total prey, 4.6% were spiders. In order to estimate the effect of F. polyctena within their hunting area on the spider population, the spiders' population density was studied inside and outside the hunting area. Samples of the forest floor were taken, spider webs were counted and pitfall traps were used. No significant difference was found in density or composition of the spider fauna inside and outside the hunting area.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.