An isoelectric variant of the 150,000-dalton neurofilament polypeptide. Evidence that phosphorylation state affects its association with the filament

A soluble isoelectric variant of the 150,000-dalton neurofilament protein was isolated from bovine brain by treating a partially purified filament preparation with a low-ionic-strength high-pH buffer. The protein (S150) had similar peptide maps to the neurofilament component of the same molecular weight (NF150) and was recognized by a polyclonal antibody made against the NF150 polypeptide. However, only half the anti-NF150 activity could be removed with the S150 protein. In addition, the S150 protein had a higher isoelectric point than the NF150 protein. Phosphate analysis indicated that the S150 protein was considerably lessened in phosphate content, which could account for the higher isoelectric point of the protein. It appears, therefore, that the S150 protein may be a precursor of NF150 or the result of phosphatase activity during the isolation procedure. Assembly studies showed that the S150 protein, unlike the NF150 protein, could not assemble with the 70-kDa neurofilament protein, indicating that the phosphate groups which were removed are important in the association of this protein to the neurofilament. When filaments containing all three triplet neurofilament polypeptides or those composed of the 70- and 150-kDa neurofilament proteins were subjected to acid phosphatase, a soluble fraction was obtained, which contained isoelectric variants with higher pI values than the NF150 polypeptide. Only unmodified NF150 protein was found in the insoluble fraction. These results support the argument that removal of phosphate groups results in the dissociation of this protein from the filament.     

Fluorescent inhibitors of a cell surface protease used to locate leukaemia cells in kidney sections

Guanidinobenzoatase is a trypsin-like protease on the surface of cells capable of migration, for example leukaemia cells. We have used a number of fluorescent probes that are competitive inhibitors of guanidinobenzoatase to locate leukaemia cells in resin sections of kidney tissue obtained from leukaemic rats. We have demonstrated how this competitive inhibition system can be used to direct desired molecules (such as cytotoxic drugs) to these cells and to monitor the arrival of such compounds at the active site of guanidinobenzoatase. The principles developed in this study could equally well be applied to other enzymes on other cells provided suitable competitive inhibitors were designed. The presence of an enzyme on the surface of a cell can be used to direct molecules to that cell provided that these molecules contain a functional group that acts as an inhibitor for the chosen enzyme.     

Enhanced binding of radioligands to receptors of γ-aminobutyric acid and benzodiazepine by a new anticonvulsive agent,LY81067

A diaryltriazine, LY81067, effectively protects against pentylenetetrazole- and picrotoxin-induced convulsions in mice, with ED₅₀ values of 5.7 and 5.8 mg/kg i.p., respectively. LY81067 enhances the binding of both ³H-GABA and ³H-flunitrazepam to specific sites in rat brain membranes. The degree of enhancement by LY81067 varies from one brain region to another and is different for the binding of ³H-GABA and ³H-flunitrazepam. In cortical membranes, LY81067 increases the affinity of ³H-GABA for both high and low affinity sites and increases the number of sites. LY81067 increases the affinity of ³H-flunitrazepam for its binding sites without greatly increasing the number of sites. Like the pyrazolopyridines, the enhancement of ³H-flunitrazepam binding by LY81067 is dependent on chloride or related anions and is reversed by picrotoxin, suggesting that LY81067 exerts its anticonvulsant effects by binding to or near picrotoxin binding sites. The differential effects of LY81067 on the enhancements of ³H-GABA and ³H-flunitrazepam binding in several brain regions suggest extensive multiplicity of GABA/benzodiazepine/picrotoxin/anioin receptor complexes.     

Enhanced natriuretic effect of ADH in rats loaded with NaCl

ADH at doses 20 μU/100 g and 100 μU/100 g or 20 μU/100 g and 200 μU/100 g was injected intravenously into pentobarbital and alcohol anaesthetized rats loaded with either water or isotonic solution consisting of NaCl, glucose and ethanol. At the dose of 20 μU/100 g ADH retained water in both water and NaCl loaded animals. At high doses 100 μU/100 g in NaCl loaded rats and 200 μU/100 g in water loaded rats, ADH retained water and increased the renal excretion of Na⁺. That the natriuretic effect of ADH at the dose 20 μU/100 g was enhanced in NaCl loaded rats suggests that ADH is probably important in the regulation of Na⁺ content in the body when it is loaded with NaCl.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

Changes in Muscarinic Binding in Distant Brain Regions Showing Neuronal Losses After Folic Acid Injections into the Substantia Innominata

Injection of folic acid (FA) into the nucleus substantia innominata (NSI) was found to decrease [3H]quinuclidinyl benzilate ([3H]QNB) binding in the frontal cortex, pyriform cortex, amygdala, and the NSI itself without changing the KD. Binding in the thalamus, caudate nucleus, hippocampus, and substantia nigra was not affected. [3H]Flunitrazepam binding was unchanged in all eight regions studied. Previous work indicates FA injections into the NSI produce epileptiform activity and cause loss of GABAergic and possibly other neurons in the frontal and pyriform cortices, the amygdala, and thalamus. The reductions of [3H]QNB binding in the first three of these regions are interpreted as indicating that many of the neurons lost are cholinoceptive, a finding that supports the previous hypothesis that activation of cholinergic projections from the NSI is an important part of the mechanism of cell loss in these regions.     

Respiratory Chain of a Pathogenic Fungus, Microsporum gypseum: Effect of the Antifungal Agent Pyrrolnitrin

Pyrrolnitrin has been reported to inhibit Bacillus megaterium primarily by forming complexes with phospholipids and to block electron transfer of Saccharomyces cerevisiae between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. We found that pyrrolnitrin inhibited respiration of conidia of Microsporum gypseum. In mitochondrial preparations, pyrrolnitrin strongly inhibited respiration and the rotenone-sensitive NADH-cytochrome c reductase. The rotenone-insensitive NADH-cytochrome c reductase, the succinate-cytochrome c reductase, and the reduction of dichlorophenolindophenol by either NADH or succinate were inhibited to a lesser extent. However, the activity of cytochrome oxidase was not affected by pyrrolnitrin. The extent of reduction of flavoproteins by NADH and succinate, measured at 465 - 510 nm, was unaltered; however, the reduction of cytochrome b, measured at 560 - 575 nm, was partially inhibited by pyrrolnitrin. The level of totally reduced cytochrome b was restored with antimycin A. We, therefore, concluded that the primary site of action of this antifungal antibiotic is to block electron transfer between the flavoprotein of the NADH-dehydrogenase and cytochrome b segment of the respiratory chain of M. gypseum.