Blocking action of Nephila clavata spider toxin on ionic currents activated by glutamate and its agonists in isolated hippocampal neurons

The blocking action ofNephila clavata spider neurotoxin, or JSTX, on ionic currents activated by L-glutamate and its agonists when applied to the membrane of neurons isolated from the rat hippocampus was investigated using a concentration clamp technique. Crude JSTX venom was found to block L-glutamate-, quisqualate, and kainate-activated ionic currents induced by activating non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Following the effects of JSTX, ionic currents activated by L-glutamate and its agonists declined to 34–36% of their initial value with no recovery during JSTX washout. An active fraction of JSTX at concentrations of 10^–4–10^–5 produced almost total but partially reversible blockade of ionic currents. The action of JSTX became less effective during depolarization. The concentration dependence of JSTX-induced blockade of kainate-activated ionic currents was investigated and the velocity constants of interaction between the toxin and glutamate receptors obtained. It is postulated that JSTX interacts with chemically-operated non-NMDA ionic channels, blocking their transition into a number of their possible open states.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 152–160, March–April, 1989.     

Role of MgATP and MgADP in the cross-bridge kinetics in chemically skinned rabbit psoas fibers. Study of a fast exponential process (C)

The role of the substrate (MgATP) and product (MgADP) molecules in cross-bridge kinetics is investigated by small amplitude length oscillations (peak to peak: 3 nm/cross-bridge) and by following amplitude change and phase shift in tension time courses. The range of discrete frequencies used for this investigation is 0.25-250 Hz, which corresponds to 0.6-600 ms in time domain. This report investigates the identity of the high frequency exponential advance (process C), which is equivalent to "phase 2" of step analysis. The experiments are performed in maximally activated (pCa 4.5-5.0) single fibers from chemically skinned rabbit psoas fibers at 20 degrees C and at the ionic strength 195 mM. The rate constant 2 pi c deduced from process (C) increases and saturates hyperbolically with an increase in MgATP concentration, whereas the same rate constant decreases monotonically with an increase in MgADP concentration. The effects of MgATP and MgADP are opposite in all respects we have studied. These observations are consistent with a cross-bridge scheme in which MgATP and MgADP are in rapid equilibria with rigorlike cross-bridges, and they compete for the substrate site on myosin heads. From our measurements, the association constants are found to be 1.4 mM-1 for MgATP and 2.8 mM-1 for MgADP. We further deduced that the composite second order rate constant of MgATP binding to cross-bridges and subsequent isomerization/dissociation reaction to be 0.57 x 10(6)M-1s-1.     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.     

Flavonoids inhibit the expression of heat shock proteins

Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetin and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DM cells. Quercetin inhibited the induction of hsp70 at the level of mRNA accumulation. This is the first report to describe the inhibition of hsp expression by reagents.     

Identification of genes involved in growth autonomy of hematopoietic cells by analysis of factor-independent mutants

The factor-dependent myeloid precursor cell line D35 mutates spontaneously at a frequency greater than 2.4 x 10(-7) to growth factor autonomy. This frequency could be increased at least 20-fold by retrovirus insertional mutagenesis. The isolation and characterization of factor-independent mutants allowed the identification of genes involved in growth autonomy. Mutants could be subdivided into two sets: those that secreted a stimulating factor (10/11) and those that did not (1/11). In one case, the factor released was distinct from previously characterized growth factors. In most mutants (6/9), the activation of a growth factor gene was associated with rearrangement that could be attributed to the insertion of a transposable-like element either 5' or 3' of the factor coding region in all cases examined, excluding oncogene involvement. All factor-independent mutants were tumorigenic, consistent with the hypothesis that growth-factor independence initiated by aberrant growth factor gene activation is an important and early step in tumorigenesis.     

Effects of medicinal plant extracts from Chinese herbal medicines on the mutagenic activity of benzo[a]pyrene

The effects of medicinal plants on the mutagenicity of benzo[a]pyrene were studied with Salmonella typhimurium tester strains. The chosen medicinal plants are very frequently used as Chinese herbal medicines. Each medicinal plant was extracted with hot water, which is similar to the method used in Chinese medicinal treatment. Cinnamomi cortex, Rhei rhizoma, Scutellariae radix and Rehmanniae radix were found to decrease the mutagenic activity of benzo[a]pyrene. Atractylodis rhizoma also reduced the mutagenicity of benzo[a]pyrene, but this was not certain, because it showed a killing effect on the cell survival test. Bupleuri radix and Aurantii nobilis pericarpium had an enhancing effect, but then neither of these extracts is itself mutagenic. Each medicinal plant extract showed a different effect on the mutagenicity of benzo[a]pyrene. These effects were classified into 5 types: (I) decreasing effect, (II) killing effect, (III) enhancing effect, (IV) enhancing and decreasing effect and (V) inactive.     

U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells.

The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.