Kinetic Effects of Terbium Ions on Muscarinic Acetylcholine Receptors of Murine Neuroblastoma Cells

Preincubation of murine neuroblastoma cells (clone N1E-115) with terbium chloride resulted in a significant potentiation of carbachol-mediated increase in cyclic GMP formation. This effect was accompanied by a shift of the peak response from 30 s to 120 s and a 6-fold decrease in carbachol concentration producing half-maximal responses, in addition to a significant increase in the Hill coefficient. Terbium ions also caused a significant decrease in the affinity and an increase in the maximum binding of [3H]quinuclidinyl benzilate to muscarinic receptors, the change in affinity being mainly due to a decrease in the association rate. Preincubation of cells with 1 mM carbachol for 4 h (the desensitized state of the muscarinic receptor) resulted in a decrease in the ability of terbium to alter [3H]quinuclidinyl benzilate binding. The effects of terbium reported here might be due to its affecting muscarinic receptor-effector coupling, which is considered to be lost upon receptor desensitization.     

Molecular organization of paramyosin in the core of molluscan thick filaments

An arrangement of paramyosin molecules in the polar part of molluscan thick filaments is proposed which accounts for the X-ray diffraction pattern of the smooth adductor muscle (other than the part ascribed to actin) and for the appearance of separated filaments in the electron microscope. The proposed structure is based on the PI arrangement of Cohen et al. (1971), and contains sets of parallel, equidistant molecules with successive molecules displaced along the molecular axis by 72 nm, which we call PI sheets. Every molecule belongs to two PI sheets which are nearly perpendicular. This array is not propagated throughout the filament, but is sheared periodically in the direction of the molecular (filament) axis by 2/5 X 72 nm. The shear occurs along parallel equidistant planes which are inclined to the PI sheets. The analysis of the X-ray data has been made possible by concentrating on those patterns from filaments in which the two sets of PI sheets appear to be mutually perpendicular, a condition brought about by bathing the muscle in aqueous acetone. In one set, there are four intermolecular spaces between shear planes (this appears to be true at least for the smooth adductors of Ostrea edulis, Crassostrea angulata and Mercenaria mercenaria). In the other set, the number varies with species and probably lies between eight and ten in the first two and appears to be six in the last named species. The known paracrystalline nature of paramyosin filaments suggests that this number, though dominant in one species, is not exactly constant.     

Inhibition of in vitro germination and spherulation ofCoccidioides immitis byAllium sativum

An aqueous extract of a dehydrated garlic preparation with uniform consistency inhibited all eight clinical isolates of the dimorphic fungus,Coccidioides immitis. The inhibitory and lethic concentrations were in the range of 3.12–6.25 mg/ml for both the saprophytic (mold) and parasitic (spherule) forms ofC. immitis. At 6.25-mg/ml concentration, the organism lost its viability within 6 h. The conversion of arthroconidia into spherules in a chemically defined liquid medium was prevented by garlic extract diluted to 1:320 (3.12 mg/ml). The data indicate that components of garlic readily inhibited the in vitro germination and spherulation of this medically important dimorphic fungus.     

Effect of dose schedule of vitamin E and hydroxethylruticide on intestinal toxicity induced by adriamycin

CDF1 mice receiving Adriamycin, 12 mg/kg IP develop a toxic GI mucositis. The mean survival in CDF1 mice after Adriamycin injection was found to be 6.5 +/- 2.0 weeks and could be increased by alcohol or acetate Vitamin E pretreatment (with 2 g/kg qDx7d) to 22.06 +/- 12.3 weeks or by treatment with Venoruton after Adriamycin (qDx7 with 1.5 g/kg) to 23.7 +/- 12.7 weeks. Other schedules were ineffective or harmful. The ability of Venoruton to enhance survival when given after Adriamycin encouraged us to proceed to tumor bearing mice. The maximum survival with CDF1 mice bearing 5 X 10(6) L1210 cells was 1 +/- 0.2 week which could be increased to 2.17 +/- 0.8 weeks with optimal dose Adriamycin (10 mg/kg). Optimum survival with Venoruton and a single dose of Adriamycin was 2.45 +/- 0.91 weeks with Venoruton, 1.5 g, qd X 14, and 12 mg/kg Adriamycin. Treatment of L1210 bearing mice with Adriamycin, 10 mg/kg on days 1 and 8, yielded a survival of 2.23 +/- 0.7 weeks. An equitoxic regimen of Adriamycin, 11 mg/kg on days 1 and 9, plus Venoruton, 1.5 g, qd X 14, increased survival 30% to 3.08 +/- 2.9 weeks. Venoruton is a promising agent to increase the therapeutic index of Adriamycin.     

The effect of temperature on the egg incubation period of Capnia bifrons (Plecoptera: Capniidae) from Windermere (English Lake District)

Gravid females of Capnia bifrons (Newman) from Windermere (English Lake District) were almost completely ovoviviparous, the eggs hatching within 15 min after oviposition in the water. When kept in the laboratory at constant temperatures between 3.8 and 19.8°C, few females survived to lay eggs at temperatures above 12.1°C. The relationship between air temperature (T°C) and the egg incubation period (Y days between fertilisation and oviposition) was given by the regression equation: Y = 316.4 T^−0.9996 (r²= 0.957, p < 0.001). This equation successfully predicted egg incubation periods for gravid females kept in cages in the field.
Comparisons with similar studies on four non-ovoviviparous species of Plecoptera showed that egg development was rarely more rapid in C. bifrons . It was also shown that the hypothesis of ovoviviparity being an adaptation to combat low water temperatures could be rejected for C. bifrons from Windermere.     

Evidence that 2-allyl-2-isopropylacetamide, phenobarbital and 3,5-diethoxycarbonyl-1,4-dihydrocollidine induce the same cytochrome P450 mRNA in chick embryo liver

The induction of cytochrome P450 in chick embryo liver has been studied using three different porphyrinogenic drugs, 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and phenobarbital. Pulse-labelling studies have shown that for each drug the cytochrome P450 synthesized either in ovo or in a wheat germ translation system reacted immunologically with antibody raised against the purified 2-allyl-2-isopropylacetamide-induced enzyme (Mr = 50000). To investigate whether this is due to the three drugs inducing the same protein or different proteins with common immunological determinants, nucleic acid hybridization studies have been carried out using a recently characterised 2-allyl-2-isopropylacetamide-induced cytochrome P450 cloned cDNA probe [Brooker, J. D. et al. (1982) Eur. J. Biochem. 129, 325-333]. It has been shown that the mRNA induced by each drug hybridizes with this probe and all are of similar size. The melting profile of the mRNA . cDNA hybrids indicates that the mRNAs induced by the three drugs have at least 98% homology with the cDNA probe. Restriction endonuclease digestions of total chick embryo genomal DNA and a chick cytochrome P450 genomal clone indicates that the cytochrome P450 gene homologous with the cDNA probe is represented in the genome only once. These results strongly suggest that the three drugs cause increased levels of the same cytochrome P450 mRNA, possibly due to enhanced expression of the same gene. Results are also presented which show that other cytochrome-P450-inducing drugs, 3-methylcholanthrene, beta-naphthoflavone or pregnenolone-16 alpha-carbonitrile do not increase the level of the 2-allyl-2-isopropylacetamide-inducible mRNA but rather reduce it to a level which was lower than that of the untreated controls.     

Phosphorylation of different sites of acetyl CoA carboxylase by ATP-citrate lyase kinase and cyclic AMP-dependent protein kinase

Native acetyl CoA carboxylase was phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase and ATP-citrate lyase kinase to 1 and 0.5 mol/subunit respectively. Both protein kinases added together increased acetyl CoA carboxylase phosphorylation additively. Partial proteolysis of 32P-acetyl CoA carboxylase followed by electrophoretic analysis showed that the 32P-phosphopeptides generated from acetyl CoA carboxylase phosphorylated with lyase kinase were different from the peptides obtained from the enzyme phosphorylated by cyclic AMP-dependent protein kinase. Mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography showed that the major phosphopeptides phosphorylated by ATP-citrate lyase kinase were different from the major phosphopeptides phosphorylated by cyclic AMP-dependent protein kinase. The results suggest that at least one different site on acetyl CoA carboxylase is preferentially phosphorylated by each protein kinase.     

Influence of sewage treatment and urbanization on selection of multiple resistance in fecal coliform populations.

The fecal coliform populations found in the raw sewages and final sewage effluents of mechanical treatment plants, a long-term retention lagoon, shorter-term retention lagoons, a remote northern Canada river, and a heavily urbanized prairie river were examined for antibiotic resistance and the possession of R factors. It was determined that there was a decrease in the percentage of multiresistant fecal coliform populations in the mechanical sewage treatment plants and shorter-term retention lagoons; however, there was an increase in populations from the long-term retention lagoon. The percentage of the populations possessing transmissible R factors was constant in the mechanical treatment and shorter-term retention facilities; however, the ability to transmit was lost in 50% of the infective population of the long-term retention facility. A striking contrast was found between the populations of the remote northern Slave River and those of the urbanized Red River. Of the fecal coliforms in the Slave River, 7.1% were multiresistant, and only 0.79% possessed transmissible R factors. The Red River fecal coliform populations were 52.9% multiresistant, and 18.77% of the total population possessed transmissible R factors. The influence of urbanization and the type of sewage treatment have been shown to affect the selection and survival of multiresistant fecal coliforms and R+ fecal coliforms. Determination of other factors influencing the development and the survival of these populations is needed for rational wastewater management and water quality consideration.     

Effects of angiotensin II and dibutyryl cyclic adenosine monophosphate on phosphatidylinositol metabolism, 45Ca2+ fluxes, and aldosterone synthesis in bovine adrenal glomerulosa cells

Angiotensin II (AII) and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) both stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. AII altered 45Ca2+ fluxes and increased 32PO4 incorporation into phosphatidylinositol in these cells, whereas dibutyryl cyclic AMP did not affect either process. Neither AII nor dibutyryl cyclic AMP increased the mass of phosphatidylinositol. Both agents are known to stimulate pregnenolone synthesis. Thus, although dibutyryl cyclic AMP and AII may increase aldosterone synthesis at a common site (pregnenolone synthesis), they do so by different mechanisms. AII stimulation of phosphatidylinositol labeling by 32PO4 (the "PI effect") was blocked when cells were incubated in a medium containing both EGTA and the calcium antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxy-benzoate hydrochloride (TMB-8), suggesting a calcium requirement for the PI effect.     

Cholesteryl hemisuccinate alters membrane fluidity, angiotensin receptors, and responses in adrenal glomerulosa cells

We examined the effects of cholesteryl hemisuccinate on membrane fluidity and angiotensin II (AII) actions in bovine adrenal glomerulosa cells. Incubating cells with cholesteryl hemisuccinate decreased membrane fluidity and markedly inhibited AII binding. The effect on binding was characterized by a decrease in AII receptor number. The effects of AII on phosphatidyl inositol turnover and calcium fluxes, proposed intermediaries of AII actions on aldosterone secretion, were less impaired than AII binding by cholesteryl hemisccinate. AII stimulation of aldosterone secretion was preserved despite the decrease in AII binding after cholesteryl hemisuccinate treatment. These results indicate that AII binding can be dissociated from its effects on aldosteronogenesis by a reagent that alters membrane fluidity.