Effect of anticancer drugs on the release of interferon γ in vitro

Summary Some conventional and experimental anticancer drugs were tested for their effect on concanavalin-A-induced interferon release from rat splenocytes in vitro. When 2.5 × 10⁶ rat splenocytes/ml, stimulated with 1 µg/ml concanavalin A, were incubated with various non-cytotoxic doses of the vinca alkaloid vincristine, there was an inhibition of the release of interferon in culture supernatants. The antitumour antibiotics bleomycin and Adriamycin, alkylating agents 4-hydroperoxycyclophosphamide and mafosfamide, and the immunoactive peptides FK 156 and FK565 did not affect the release of interferon under similar conditions. However, cyclosporin A, in similar experiments, markedly inhibited the release of interferon .     

Screening Actinomycetes for Extracellular Peroxidase Activity

A diverse collection of actinomycete strains were screened for production of extracellular peroxidase activity by adapting a chemiluminescence analysis system developed for horseradish peroxidase-based enzyme-linked immunosorbent assay. Extracellular peroxidase activity was found to be common but quantitatively variable, and this rapid and sensitive screening system permitted identification of a small group of high-producing strains. A range of spectrophotometric assays were compared for the measurement of peroxidase activity in concentrated culture supernatants of two selected thermophilic streptomycetes. Of these, the peroxide-dependent oxidation of 2,4-dichlorophenol was identified as the most robust and reproducible assay for quantitative studies.     

Ricin B chain fragments expressed inEscherichia coli are able to bind free galactose in contrast to the full length polypeptide

Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced inE. coli and targeted to the periplasm by fusion to theompA orompF signal sequences. The proteins were then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such proteins produced inXenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins. When produced inE. coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced inXenopus laevis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides. However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose. The only deletion mutant observed to bind to free galactose when produced inE. coli corresponded closely to the complete domain 2 of RTB. It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility toE. coli proteases.Abbreviations RTA ricin toxin A chain - RTB ricin toxin B chain - ER endoplasmic reticulum - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - IPTG isopropyl -d-thiogalactopyranoside     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Petiolar felt-sheath of palm: A new matrix for fungal immobilization

Summary Petiolar felt-sheath of palm (Livistona chinensis) has been successfully used as a new biostructural matrix for the immobilization of fungal hyphae. The reticulated felt-sheath is made up of fibrous bundles having irregularly scattered membranous-foliose and fibrous outgrowths woven into a cohesive multi-layered mesh. Immobilization of Aspergillus niger was achieved with both spore and hyphal suspensions as the inoculum. Growth in immobilized cultures was 19% greater than in free cultures.     

Regulation of calmodulin content in synaptic plasma membranes by glucocorticoids

Synaptic plasma membranes (SPM) from the brain are known to have specific binding sites for several steroid hormones, but the mechanisms of membrane transduction of steroid signals is not understood. In this study, corticosterone was found to prevent temperature-dependent dissociation of endogenous calmodlin (CaM) from highly purified SPM from rat cerebral cortex. The steroid stabilizes Ca²⁺-dependent membrane binding of endogenous CaM (78% of total CaM), whereas Ca²⁺-independent binding of CaM (the other 22%) is not affected. The stabilization of membrane binding of endogenous CaM by corticosterone is concentration-dependent, with the maximal effect occurring at steroid concentration of 1 M. The EC₅₀ is estimated as 130 nM, which is almost identical to the K_d of specific binding of the steroid to SPM (120 nM) reported previously. The effect in stabilizing membrane binding of CaM is specific to corticosterone and other glucocorticoids (cortisol, dexamethasone and triamcinolone); gonadal steroids (17-estradiol, progesterone and testosterone) are ineffective. Furthermore, corticosterone administration in vivo (2 mg/kg, i.p.) produced a rapid increase of CaM content in SPM, occurring within 5 min after steroid injection and persisting for at least 20 min. Since CaM mediates a variety of biochemical processes in synaptic membranes, we hypothesize that the effect of glucocorticoids in promoting membrane binding of CaM may lead to a cascade of consequences in synaptic membrane function.Special issue dedicated to Dr. Sidney Ochs.     

Regulation of N-Methyl-d-aspartate receptor-mediated calcium transport and norepinephrine release in rat hippocampus synaptosomes by polyamines

The role of polyamines (PA) synthesis in NMDA receptor-mediated⁴⁵Ca²⁺ fluxes and norepinephrine release was studied in rat hippocampal synaptosomes. NMDA (50M) caused a sharp (>2-fold) transient increase in PA synthesis regulating enzyme, ornithine decarboxylase (ODC) activity with concomitant elevation in PA levels in the order putrescine>spermidine>spermine. ODC inhibitor, -difluoromethylornithine (DFMO), and NMDA antagonist, 2-amino-5-phosphonovaleric acid (D-AP5), both blocked increases in ODC activity and PA levels. Activation of NMDA receptors induced a sharp (3 to 4-fold) and quick (15 seconds) increase in⁴⁵Ca²⁺ uptake by synaptosomes within 15 seconds of exposure at 37°C. The efflux of⁴⁵Ca²⁺ and³H-norepinephrine (NE) release at 22°C from pre-loaded synaptosomes was also significantly (2 to 4-fold) enhanced by NMDA within 15 seconds. These NMDA receptor-mediated effects on calcium fluxes and NE release were blocked by NMDA receptor-antagonists (DAP-5 and MK-801) and PA synthesis inhibitor, DFMO and the DFMO inhibition nullified by exogenous putrescine. These observations establish that ODC/PA cascade play an important role in transduction of excitatory amino acid mediated signals at NMDA receptors.Special issue dedicated to Dr. Sidney Ochs.     

Analysis of γ-Aminobutyric AcidA Receptor Subunits in the Mouse Cochlea by Means of the Polymerase Chain Reaction

Abstract: Unlike 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces consistent decreases in levels of striatal dopamine (DA) with considerably smaller and more variable effects on mouse brain levels of serotonin (5-HT) and norepinephrine (NE), a novel amine-substituted MPTP analogue, 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH₂-MPTP), administered in a standard mouse dosing paradigm for MPTP (20 mg/kg X 4) did not affect striatal DA but led to marked reductions (60–70%) in levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and NE measured in frontal cortex and hippocampus 1 week after treatment. Another 2'-substituted MPTP analogue, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine, affected cortical and hippocampal 5-HT, 5-HIAA, and NE only minimally, while markedly reducing the DA content in striatum (90%), thus indicating that the substituent (-NH₂ versus -CH₃) at the 2'position is important for the differential effects of these MPTP analogues. In a replication study with a 3-week end point, hippocampal and cortical 5-HT, 5-HIAA, and NE levels remained depressed with no indication of recovery. These results suggest that 2'-NH₂-MPTP may be a novel, regionally selective neurotoxin for serotonergic and norad-renergic nerve terminals.