Reversal by EGTA of the enhanced secretory responsiveness of mast cells due to treatment with ouabain

The effect of EGTA on the enhancement by ouabain of compound 48/80-induced secretion from mast cells was compared with the effect on the Na(+)-K+ pump activity. The time-dependent secretory enhancement by ouabain was blocked by addition of EGTA to the cell suspension concomitantly with the addition of ouabain, and EGTA caused a large increase in the pump activity. Addition of 10 microM EGTA to ouabain-treated cells stopped but did not reverse the enhancement. The experiments show that the effect of ouabain was due to changes in a calcium pool utilized in compound 48/80-induced secretion following changes in the Na+,K+ pump activity.     

Potassium channels in basolateral membrane vesicles from pars convoluta of rabbit proximal tubule

The characteristics of 86Rb+ fluxes through conductive channels in basolateral-membrane vesicles isolated from pars convoluta of rabbit proximal tubule were investigated. In KCl loaded vesicles a transient accumulation of 86Rb+ was observed which was inhibited by BaCl2. The accumulation was driven by an electrical diffusion potential, as shown in experiments using membrane vesicles loaded with Li2SO4 and an outwardly directed Li+ gradient established with a Li(+)-ionophore. The vesicles containing the channel showed a cation selectivity with the order K+ = Rb+ much greater than Li+ greater than or equal to Na+ greater than choline+. The 86Rb+ flux was dependent on intravesicular Ca2+. Increasing concentrations of Ca2+ gradually decreased the 86Rb+ uptake.     

Rapid purification of cobalamin-binding proteins using immobilized aminopropylcobalamin

Aminopropylcobalamin (AP-Cbl), prepared from 3-chloropropylamine and cob(I)alamin, was immobilized on CNBr-activated Sephacryl beads. The product, Sephacryl-aminopropylcobalamin, contained ca. 1 μmol of AP-Cbl/ml of beads. Cobalamin-binding proteins in biological fluids were adsorbed selectively and quantitatively by Sephacryl-aminopropylcobalamin. After being washed to remove extraneous protein, the beads were photoirradiated to release the cobalamin-binding proteins as their aquacobalamin complexes. The latter could be converted to labeled cyanocobalamin complexes by treatment with [¹⁴C]KCN. The efficacy of this affinity chromatographic method is illustrated by the purification to near homogeneity and in high yield of three representative proteins: transcobalamin II from rabbit serum, intrinsic factor from human gastric juice, and R binder from human saliva.     

Folate polyglutamate and monoglutamate accumulation in normal and SV40-transformed human fibroblasts

Folate polyglutamate and monoglutamate accumulation was measured in normal diploid and SV40-transformed human fibroblasts by Sephadex G-10 gel filtration chromatography. The cells were first depleted of folates and then provided with limiting amounts of [3H]-folic acid in order that the cells would accumulate only forms of folate necessary for proliferation. Both the normal and the transformed cells accumulated monoglutamate and polyglutamate forms, but by 72 hours of labeling the transformed cells contained 3-10 times more polyglutamate than the normal cells. The growth rates for the normal and transformed cells were similar at this limiting folic acid concentration. Thus, if folate polyglutamates are more important for the proliferation of SV40-transformed cells than the normal cells, then inhibition of polyglutamate formation may be an important potential target for chemotherapy.     

Chromosome numbers and taxonomy in Cryptocoryne (Araceae). II

Chromosome numbers for 38 species of Cryptocoryne are reported, 16 of which have not been reported earlier. About 90% of the species of Cryptocoryne have now been investigated cytologically. On the basis of chromosome numbers, morphology, and distribution, it is possible to distinguish some 24 groups within the species investigated. The chromosome numbers represent a heteroploid series which is based on the (secondary) base numbers 10, 11, 14, 15, 17, and 18. Evidence is given that 2n = 36 is the more primitive one. Evolution on the chromosome level has most likely gone in the direction of a reduction in numbers. Two new species, Cryptocoryne amicorum De Wit & N. Jacobsen and C. keei N. Jacobsen, are described.     

A mouse cell line, which is unprotected by interferon against lytic virus infection, lacks ribonuclease F activity

A mouse cell line, NIH 3T3, does not respond to some of the activities of interferon. Even after treatment with high concentrations of interferon the replication of lytic viruses, such as encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) is not inhibited in these cells. In contrast, interferon treatment of these same cells results in the inhibition of Moloney murine leukemia virus (MMuLV) production. We have analyzed enzymatic pathways which are induced by interferon in these cells. After interferon treatment, the level of the (2'-5')oligoadenylate [(2'-5)An] synthetase activity and the phosphorylation of the 67000-dalton protein (P1) are enhanced in NIH 3T3 cells to approximately the same level as interferon-sensitive mouse L-cells. Moreover, NIH 3T3 and L-cells, contain approximately the same levels of enzymes which inactivate (2'-5')An. Both exogenously added (2'-5')A3 or double-stranded RNA (dsRNA) failed to inhibit protein synthesis in NIH 3T3 extracts even though they were potent inhibitors of L-cell extract-directed protein synthesis. Direct measurements of the (2'-5')An-dependent ribonuclease F (RNase F) failed to detect such activity in NIH 3T3 cells. Our results, therefore, suggest that the presence of RNase F activity is necessary for the interferon-induced antiviral activity against EMCV and against VSV. The induction of protein kinase activity by interferon treatment of NIH 3T3 cells appears to have no direct effect on EMCV and VSV replication.     

Cryptofluorescent analogs of cobalamin coenzymes: synthesis and characterization.

Upper-axial (beta-position) ligand analogs of the B12 coenzymes 5'-deoxy-5'-adenosylcob(III)alamin and methylcob(III)alamin have been synthesized by reaction of the 5'-chloro-5'-deoxy derivatives of fluorescent nucleosides (1,N6-ethenoadenosine, formycin, 2-amino-nebularine, and 2,6-diaminonebularine) and a fluorescent alkyl halide (dansylamidopropyl chloride) with cob(I)alamin. These analogs were nonfluorescent, but fluorescent products could be generated by photolysis or cyanolysis of the carbon-cobalt bonds. Under anaerobic and aerobic conditions, the major fluorescent photolysis products of 1,N6-ethenoadenosylcob(III)alamin were 1,N6-etheno-5',8-cyclic-5'-deoxyadenosine, and the 5'-aldehyde of 1,N6-ethenoadenosine, respectively. The cryptofluorescent property of these analogs was utilized to follow the kinetics of aerobic photolysis. First-order rate constants determined by this method were comparable to those obtained spectrophotometrically [via appearance of of aquacob(III)alamin]. Pseudo-first-order rate constants determined fluorometrically for the cyanolysis (at 25 degrees C) of 1,N6-ethenoadenosylcob(III)alamin, 2,6-diaminonebularinylcob(III)alamin, 2-aminonebularinylcob(III)alamin and formycinylcob(III)alamin were 5.8 X 10(-2), 2 X 10(-2), 1.8 X 10(-2), and 3 X 10(-5) min-1, respectively; values in good agreement were obtained spectrophotometrically (via appearance of dicyanocobalamin). Dansylamidopropylcob(III)alamin was stable in the presence of cyanide. The nucleoside alpha-ribazole is fluorescent in the free state but nonfluorescent when present as the lower axial (alpha-position) ligand in cobalamin coenzymes. Thus fluorescence of ligands in both the alpha- and beta-positions of cobalamins is quenched, probably as a result of intramolecular energy transfer between the ligands and the nonfluorescent corrinoid.     

Reversion to methionine independence by malignant rat and SV40-transformed human fibroblasts.

Although many lines of malignant and transformed cells are unable to grow in folate- and cobalamin-supplemented medium in which methionine is replaced by homocysteine its immediate metabolic precursor, rare cells from these lines regained the normal ability to grow under these conditions. Six revertant lines, one from Walker-256 rat breast carcinoma cells and five from SV40-transformed human fibroblasts, have been characterized with regard to growth and three measures of methionine biosynthetic capacity: methionine synthetase and methylenetetrahydrofolate reductase activities in cell extracts, and uptake of label from [5-¹⁴C]methyltetrahydrofolate by intact cells. When all three measures of methionine biosynthetic capacity were considered, two revertants isolated from SV40-transformed cells had regained the ability to grow like normal cells in homocysteine medium without substantial changes in these measures. Increased methionine biosynthesis thus is not a prerequisite to reversion of the methionine auxotrophy present in the transformed parental lines.     

Transplantation of rabbit kidneys perfused with glycerol solutions at 10 degrees C.

Experiments are described in which rabbit kidneys were perfused with solutions containing 2, 3, or 4 m glycerol and then were autografted in order to measure any functional impairment. The basic perfusate contained Haemaccel (17.5 g/litre) and mannitol (111 mm) and had a total osmolality of 400 mosmol/kg. The perfusion temperature was 10 °C. Glycerol concentration was slowly increased at a rate of 30 mm min^?1 to 2, 3, or 4 m, was held at the maximum concentration for 30 min, and was then decreased to < 0.1 m at 30 mm min^?1. An immediate contralateral nephrectomy was carried out, and the function of the perfused kidney was assessed by serial measurements of blood urea and serum creatinine, by the determination of 24-hr endogenous creatinine clearance at 1 month and by histological examination. It was found that a concentration of 3 m glycerol was readily tolerated.