TRPC channels: integrators of multiple cellular signals.

TRPC channels are ubiquitously expressed among cell types and mediate signals in response to phospholipase C (PLC)-coupled receptors. TRPC channels function as integrators of multiple signals resulting from receptor-induced PLC activation, which catalyzes the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 depletes Ca2+ stores and TRPC3 channels can be activated by store-depletion. InsP3 also activates the InsP3 receptor, which may undergo direct interactions with the TRPC3 channel, perhaps mediating store-dependence. The other PLC product, DAG, has a direct non-PKC-dependent activating role on TRPC3 channels likely by direct binding. DAG also has profound effects on the TRPC3 channel through PKC. Thus PKC is a powerful inhibitor of most TRPC channels and DAG is a dual regulator of the TRPC3 channel. PLC-mediated DAG results in rapid channel opening followed later by a slower DAG-induced PKC-mediated deactivation of the channel. The decreased level of PIP2 from PLC activation also has an important modifying action on TRPC3 channels. Thus, the TRPC3 channel and PLCgamma form an intermolecular PH domain that has high specificity for binding PIP2. This interaction allows the channel to be retained within the plasma membrane, a further operational control factor for TRPC3. As nonselective cation channels, TRPC channel opening results in the entry of both Na+ and Ca2+ ions. Thus, while they may mediate Ca2+ entry signals, TRPC channels are also powerful modifiers of membrane potential.

Coupled ion movement underlies rectification in an inward-rectifier K+ channel.

We studied block of the internal pore of the ROMK1 inward-rectifier K+ channel by Mg2+ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K+ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zdelta values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K+. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zdelta), varied as a function of the concentration of extracellular K+. These results suggest that there is energetic coupling between the binding of blocking and permeating (K+) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K+ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K+.

Tuning the voltage dependence of tetraethylammonium block with permeant ions in an inward-rectifier K+ channel.

To understand the role of permeating ions in determining blocking ion-induced rectification, we examined block of the ROMK1 inward-rectifier K+ channel by intracellular tetraethylammonium in the presence of various alkali metal ions in both the extra- and intracellular solutions. We found that the channel exhibits different degrees of rectification when different alkali metal ions (all at 100 mM) are present in the extra- and intracellular solution. A quantitative analysis shows that an external ion site in the ROMK1 pore binds various alkali metal ions (Na+, K+, Rb+, and Cs+) with different affinities, which can in turn be altered by the binding of different permeating ions at an internal site through a nonelectrostatic mechanism. Consequently, the external site is saturated to a different level under the various ionic conditions. Since rectification is determined by the movement of all energetically coupled ions in the transmembrane electrical field along the pore, different degrees of rectification are observed in various combinations of extra- and intracellular permeant ions. Furthermore, the external and internal ion-binding sites in the ROMK1 pore appear to have different ion selectivity: the external site selects strongly against the smaller Na+, but only modestly among the three larger ions, whereas the internal site interacts quite differently with the larger K+ and Rb+ ions.

Molecular analysis of a new cytoplasmic male sterile genotype in sunflower.

Mitochondrial DNA from 1 fertile and 6 cytoplasmic male sterile (CMS) sunflower genotypes was studied. The CMS genotypes had been obtained either by specific crosses between different Helianthus species or by mutagenesis. CMS-associated restriction fragment length polymorphisms (RFLPs) were found in the vicinity of the atpA locus, generated by various restriction enzymes. The organization of the mitochondrial genes 26S rRNA, 18S + 5S rRNA and coxII was investigated by Southern blot analysis. These genes have similar structures in fertile and all studied sterile sources. Using the atpA probe, 5 from the 6 investigated CMS genotypes showed identical hybridization patterns to the Petiolaris CMS line, which is used in all commercial sunflower hybrids. Only 1 cytoplasm derived from an open pollination of Helianthus annuus ssp. texanus, known as ANT1, contained a unique mitochondrial DNA fragment, which is distinguishable from the fertile and sterile Petiolaris genotypes and from all investigated CMS genotypes. Male fertility restoration and male sterility maintenance of the ANT1 line are different from the Petiolaris CMS system, which is a confirmation that a novel CMS genotype in sunflower has been identified.

Dip patch clamp currents suggest electrodiffusive transport of the polyelectrolyte DNA through lipid bilayers.

Planar lipid bilayers formed from monolayers of diphytanoyl lecithin (DPhL) were found to interact with plasmid DNA (5.6 kbp; M(r) = 3.7 x 10(6)) leading to an increase in the conductance of the membrane. The association of DNA with a lipid bilayer greatly facilitates the transport of the small ions of the main salt KCl. The appearance of long-lived current levels, for instance, of 27.6 pA at Vm = +60 mV membrane voltage, where the actual contact (adsorption) is electrophoretically enhanced, suggests a locally conductive DNA/lipid interaction zone where parts of the DNA strand may be transiently inserted in the bilayer, leaving other parts of the DNA probably protruding out from the outer surface of the bilayer. At Vm = -60 mV, where DNA can be electrophoretically moved away from the membrane, the membrane current is practically zero. This current asymmetry is initially also observed at higher voltages, for instance at 200 mV. However, if the voltage sign (Vm = +200 mV) is changed after a transient positive current (approximately 15 pA) was observed, there is also now (at Vm = -200 mV) a finite negative current at the negative membrane voltage. Thus, it appears that at Vm = +200 mV the adsorbed parts of the polyelectrolyte DNA are not only transiently inserted in, but actually also electrophoretically pulled through, the porous zones onto the other membrane side leaving the bilayer structure basically intact. These data provide direct electric evidence for the electrophoretic transport of a highly charged and hydrated macromolecule, probably together with the associated gegen-ions, through the thin hydrophobic film of the lipid bilayer.

Lysine-orotate potentiates the toxicity of an extract of the mushroom Amanita phalloides.

Upon simultaneous administration lysine-orotate increases (about 40-fold) the toxicity in mice of a crude Amanita phalloides extract. This effect, though less prominent, is also observed if these two compounds are injected 1 hr apart. The potentiating effect of lysine-orotate is dose dependent and neither L-lysine nor orotic acid exert any effect on the toxicity of the crude A. phalloides extract. Lysine-orotate increases the toxicity of amatoxins (alpha-amanitin) only, not affecting the toxicity of phalloidins. Thin layer chromatography on silica gel plates and column chromatography on Sephadex LH-20 has proven the formation of a relatively stable complex of amanitin and lysine-orotate. The results demonstrate that lysine-orotate should not be used as a hepatoprotective agent in cases of Amanita intoxication.

Survival of lung cancer patients treated with BCG and/or a soluble BCG fraction (F70) after surgery, radiotherapy and chemotherapy.

243 lung cancer patients treated with routine therapy (surgery 54, radiotherapy 46 and chemotherapy 143) in Plovdiv's District Oncologic Dispensary (PDOD) were submitted to an additional therapy with BCG or with its soluble fraction F70. The survival rate of these patients was compared with the survival rate of 305 lung cancer patients following routine therapy alone (surgery 78, radiotherapy 113 and chemotherapy 114). The two groups were not randomized. Between patients treated and non-treated with BCG(F70) a significant increase of the survival rate was found for patients submitted to BCG(F70) treatment after surgery or radiotherapy in the 2nd and 3rd year survival period and for patients submitted to BCG(F70) treatment after chemotherapy -- in the 1st year survival period of patients with limited disease only. The effect of the additional BCG(F70) treatment was less dependent on the preceding routine therapy than upon the clinical stage of the disease at the moment of BCG(F70) treatment beginning. More salient results were observed in patients with undifferentiated histological forms.

BCG immunotherapy of lung cancer in a district oncology dispensary. I. Study of 860 patients with histologic diagnosis.

The investigation refers to lung cancer patients registered in an oncology dispensary from 1965 to 1975. The conventional treatment (surgery, radiotherapy and chemotherapy) has covered 35.9% of all patients, whereas immunotherapy with BCG or with its soluble fraction has been applied in 46.2% of them. Immunotherapy has mainly been applied to epidermoid carcinoma and large-cell carcinoma. Comparing the survival of immunotherapy treated with non-immunotherapy treated patients it was found that in Stage I-II group the difference in favor of immunotherapy was significant in the second year of observation and in Stage III group a significant difference was found in all observation periods (1-5 years). In Stage I-II the 5-year survival rate of immunotherapy treated patients (13.2%) was similar to the survival rate of operated patients (13.7%). Immunotherapy in large cell carcinoma showed significantly better results than immunotherapy in epidermoid carcinoma. Immunotherapy with BCG and F70 was applied as an independent treatment of lung cancer. When applied to patients primarily operated, radiation or chemotherapy treated, the survival was significantly higher than the survival of patients submitted to conventional treatment or to immunotherapy separately.

Synthesis, structure and antibacterial activity of some alkyltriazenopyrazoles.

A series of bis-beta-chlorethyl-, dimethyl- and diethyltriazenopyrazoles (I-IV) were synthesized. By the method of IR spectroscopy the most probable transformation form of I was shown. The compounds were tested as antibacterial agents on a series of bacterial species. It was established that only one of them (VI) possessed a low inhibitory effect. All the rest inhibited strongly the growth of Staphylococcus aureaus 209 and E. coli 387.

Cyanopyrazoles as analogs of purine precursors--I. Inhibitory effect on purine biosynthesis de novo.

The in vitro inhibition of purine biosynthesis de novo by a series of cyanopyrazoles was studied. At concentration 1 mM trichloromethyl analogs (3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole and N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole) were found to inhibit IMP synthesis 80 and 30% respectively. GAR synthesis was inhibited at a lower degree at the same range of concentrations. The compounds demonstrated a similar pattern of inhibition of the last steps, e.g. AICAR formylation and cyclization as found on the whole pathway.

Synthesis and antitumor activity of alkyltriazenopyrazoles.

The chemical synthesis of certain mono- and bis-dialkyltriazenopyrazoles is described. In antitumor studies it was found that none of the compounds produced increase in life span (ILS) of L 1210 bearing mice or inhibition of adenocarcinoma 755 growth above the criteria established. The introduction of a second triazenogroup increases the toxicity of the compounds tested.

Cyanopyrazoles as analogs of purine precursors--II. Effects on macromolecular synthesis and cell cycle progression.

The cytotoxic and cytokinetic effects, and in vitro inhibition of macromolecular synthesis by cyanopyrazoles were studied using Friend leukemia and Ehrlich ascites tumor cells. At concentrations in the range of 2.5 mM to 50 microM analog 3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole (I) was highly cytotoxic and completely inhibited thymidine, uridine and leucine incorporation into macromolecular material. 24 hr incubation of FL cells with cytostatic concentrations of compound I (in the range of 2 to 0.5 microM) resulted in an accumulation of cells in the G2 + M phase. Analogs N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole (II) and 3(5)-amino-4-cyanopyrazole (III) were not cytotoxic at concentrations up to 5 mM and did not substantially inhibit precursor incorporation into macromolecules but exhibited a cytostatic activity. These compounds caused a decrease of FL cells in the G2 + M phase and an accumulation in the S phase. Analogs I and II displayed a similar in vivo inhibitory effect on thymidine incorporation into DNA in EAT cells. The results indicate that the cytotoxicity of cyanopyrazoles correlates with their ability to inhibit precursor incorporation into macromolecular material. On the other hand, the cytostatic action of compound I is not coupled to a block of nucleic acid synthesis.

The character of growth of mycobacterium tuberculosis in vitro in dependence on peripheral human blood's components.

Blood plasma, cell mass, white blood cells (WBC) and erythrocytes were investigated for their relation to cord formation of M. tuberculosis, cultivated in blood according to Pryce's method. It was found that cord formation was related to blood cell and followed the zone of sedimentation of WBC. In lysed blood cord formation could be revealed on the whole glass surface, but disappeared if the lysed blood was filtered through Seitz filter - evidences which were accepted as indications that cord formation was dependent on WBC. However, destroying WBC in lysed blood by freezing and thawing or eliminating them by centrifugation did not disturb cord formation and addition of WBC to simple media did not result in cord formation. It was concluded that cord formation was called forth by lysed erythrocytes and this was confirmed through adding erythrocytes stromata to simple media, where cord formation appeared. The practical value of the technique applied and the eventual role of erythrocyte stromata's lipids in cord formation are discussed.

The mutagenicity of azido derivatives of monosaccharides and alcohols and of N-(3-azido-2-hydroxypropyl) derivatives of purines and pyrimidines in Arabidopsis thaliana and in Salmonella typhimurium.

The azido derivatives of alcohols (3-azido-1,2-propandiol and 1,3-diazido-2-propanol) and monosaccharides (6-azido-6-deoxy-beta-D-glucose and 6-azido-6-deoxy-beta-D-galactose), as well as the proximal mutagenic product of sodium azide metabolism beta-azido-L-alanine, exhibited a high mutagenic activity in a higher plant Arabidopsis thaliana and in Salmonella typhimurium. In contrast, 11 N-(3-azido-2-hydroxypropyl) derivatives of purines and pyrimidines (adenine, thymine, uracil, cytosine, 2-amino-6-chloropurine, 6-chloropurine, 2,6-diaminopurine, 6-methylthiopurine, 4-O-methylthymine, 4-O-methyluracil and 7-deaza-8-azaadenine) were mutagenic in the Ames assay but ineffective in the Arabidopsis mutagenicity assay.

Chick cochlear hair cell exocytosis mediated by dihydropyridine-sensitive calcium channels.

1. A semi-intact preparation of the chick basilar papilla was developed to study calcium-dependent neurotransmitter release by tall hair cells (avian equivalent of cochlear inner hair cells). 2. Tall hair cell depolarization resulted in changes in cell membrane capacitance (DeltaC(m)) that reflected cell surface area increases following synaptic vesicle exocytosis and provided a surrogate measure of neurotransmitter release. Both calcium current (I(Ca)) and DeltaC(m) were reversibly blocked by cobalt, and exhibited a similar bell-shaped dependency on voltage with a peak response around -10 mV. 3. Pharmacological agents selective for L-type calcium channels were employed to assess the role of this channel type in neurotransmitter exocytosis. Nimodipine, a dihydropyridine (DHP) antagonist, suppressed I(Ca) and blocked DeltaC(m). Conversely, the DHP agonist Bay K 8644 increased both I(Ca) and DeltaC(m) amplitude nearly 3-fold. These findings suggest that chick tall hair cell neurotransmitter release is mediated by calcium influx through L-type calcium channels.