Effect of ions on counterion fluctuation in low-molecular weight DNA dielectric dispersions

The dielectric permittivity of aqueous solutions of low-molecular weight DNA (Mr = 3.2 X 10(5) ) in the presence of MgCl2 and AgNO3 has been measured in the frequency range from 5 kHz to 30 MHz, at a temperature of 25 degrees C. The DNA concentration was 3.5 X 10(-4) M in terms of phosphate and the salt concentration was varied from 1 X 10(-5) to 2 X 10(-4) M. The dielectric results have been analyzed in terms of two contiguous dielectric dispersions, and characteristic parameters have been discussed on the basis of polyelectrolyte theories which deal with counterion fluctuation. Some molecular parameters of the DNA molecule in electrolyte solutions are estimated.     

Dependency of delta pH-relaxation across vesicular membranes on the buffering power of bulk solutions and lipids.

The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions.     

The effects of furosemide on adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate and corticosterone production stimulated by adrenocorticotropin in monolayer cultured rat adrenal cells

Furosemide has been reported to have a suppressive effect on ADH-, PTH- and adrenaline-stimulated adenosine 3':5'-cyclic monophosphate (cAMP) production, but the effect on adrenocorticotropin (ACTH) action has not yet been elucidated. In the present study, therefore, the effects of furosemide on cAMP and also on guanosine 3':5'-cyclic monophosphate (cGMP) and corticosterone, stimulated by ACTH in monolayer cultured rat adrenal cells, were investigated. The intra- and extracellular cAMP stimulated by ACTH was dose-dependently suppressed by furosemide within the concentration range of 10(-3) M to 3 X 10(-3) M, and the suppressive effect of the drug was accompanied with decreased corticosterone production. However, non-stimulated basal corticosterone production was not influenced by the drug even at 3 X 10(-3) M. A similar suppressive effect of dibutyryl cAMP-stimulated corticosterone production by 3 X 10(-3) M furosemide was observed. The intracellular cAMP bound to its binding protein in sonicated adrenal cell extract was also suppressed in a very similar dose-dependent manner to total cAMP. However, though the effect on corticosterone production was also observed when the calcium concentration in the loading medium was changed, the magnitude of the effectiveness (percent of control) was relatively constant at each calcium concentration, suggesting that furosemide may not affect the site(s) at which calcium acts. Intracellular cGMP, on the other hand, was increased by 10(-3) M to 3 X 10(-3) M of furosemide, suggesting an intensifying effect of furosemide on guanylate cyclase activity. Dibutyryl cGMP-stimulated corticosterone production was also increased at the same concentration range. These results indicated that furosemide may act not only on adenylate cyclase but also on the additional step(s) to suppress the resultant corticosterone production. In contrast to the effects of furosemide on such cAMP-mediated processes, this drug treatment appeared to enhance cGMP-mediated corticosterone production.     

Liposome formation of egg lecithin and its interaction with iodine

The sonicated dispersion of egg lecithin (phosphatidylcholine) in water forms 1:1 molecular complex with iodine, when its concentration is above 1.6 X 10(-5) M. The thermodynamic and spectrophotometric properties of this complex have been determined. The thermodynamic values are: K (25 degrees C) = 1.6 X 10(3) 1 X mol-1, delta G degrees = -18.4 KJ X mol-1, delta H degrees = -27.4 KJ X mol-1 and delta S degrees = -30.0 J X mol-1 X deg-1. The complex shows two absorption bands: one at 293 nm, which is the charge transfer band and the other at 370 nm, which is the blue shifted visible iodine band at 460 nm in water.     

Characterization of Populations of Promastigotes of Leishmania donovani1

Promastigotes of Leismania donovani cultured for either 3 or 10 days in vitro and inoculated intracardially into golden hamsters with an equal number of organisms from either population showed a 7-fold difference in infectivity when compared at both 10 to 16 days post-infection. Reproducible histochemical staining for the promastigote enzymes glucose-6-phosphate dehydrogenase (G6PDH) and peptidase after polyacrylamide gel electrophoresis showed two isoelectric variants of G6PDH (Bands 1 and 2) that displayed a 45% decrease (Band 1) and a 60% increase (Band 2) in total activity when 3- and 10-day-old promastigores were compared. Peptidase activity, present in a single band, increased 7-fold in 10-day-old promastigotes. A decrease in the lectin-induced agglutination of promastigotes by castor bean agglutinin (RCA₆₀), specific for D-galactose and N-acetyl-D-galactosamine, was seen when 3- and 10-day-old promastigotes are compared. Antisera raised against sonicated 10-day-old promastigotes showed a unique precipitin band between the antiserum and sonicated 10-day-old promastigotes not found between the antiserum and sonicated 3-day-old promastigotes.     

The effect of prostaglandins E1, E2, F1 alpha and F2 alpha on pig erythrocytes during haemolysis induced with aspirin and hypotonic NaCl solution

The effect of prostaglandins PGE1, PGE2, PGF1 alpha and PGF2 alpha was investigated on the haemolysis of pig erythrocytes induced with aspirin and hypotonic (0.119 M) NaCl solution. An inhibiting effect was observed of low concentrations (2 X 10(-5) M, 2 X 10(-4) M and 2 X 10(-3) M) of aspirin on haemolysis induced with hypotonic NaCl solution, while in a concentration of 2 X 10(-2) M aspirin itself caused haemolysis which amounted to 93% of the haemolysis induced with 0.041 M NaCl solution. No differences were observed in the degree of haemolysis inhibition in relation to the time of incubation of erythrocytes with aspirin. Aspirin concentrations from 0.035 M to 0.280 M caused slight haemolysis (9-15% of the haemolysis induced with water), the 0.560 M solution caused haemolysis corresponding to 85% of the water-induced haemolysis. None of the studied prostaglandins used in concentrations of 0.4 X 10(-3) M, 0.4 X 10(-4) M and 0.4 X 10(-5) M had any significant effect on aspirin-induced haemolysis. PGE1 and PGE2 in concentrations of 0.4 X 10(-3) M, 0.4 X 10(-4) M and 0.4 X 10(-5) M inhibited haemolysis induced with 0.119 M sodium chloride solution, and the degree of haemolysis inhibition was from 8% to 35%. Prostaglandins PGF1 alpha and PGF2 alpha in the same concentrations had no protective effect.     

The relationship between DNA damage and mutation frequency in mammalian cell lines treated with N-nitroso-N-2-fluorenylacetamide

The induction of DNA single-strand breaks in C3H10T1/2 mouse fibroblasts and Chinese hamster ovary (CHO) cells by N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA) was demonstrated by the alkaline elution technique. Without metabolic activating system (i.e., rat liver S9 fraction), N-NO-2-FAA exhibits more direct and strong damaging effects on DNA than its parent compound, 2-FAA, at equal concentration in both cell lines. To compare the DNA-damaging potency of N-NO-2-FAA with other well-known carcinogens, such as benzo[a]pyrene, 2-nitrofluorene, and N-methyl-N'-nitrosoguanidine (MNNG), the order of potency is as follows: MNNG (5 microM) greater than N-NO-2-FAA (150 microM) greater than benzo[a]pyrene (20 microM) at equitoxic concentrations, LD37, in the same cell system. Another parallel experiment indicated that N-NO-2-FAA could disrupt the superhelicity of circular plasmid DNA (pBR 322) at a dose range of 0.1-50 mM; however, a complete conversion to form III linear DNA was found at the highest concentration (50 mM). After treatment with various concentrations of N-NO-2-FAA, ouabain resistance (ouar) was induced in C3H10T1/2 cells, while both ouar and 6-thioguanine resistance (6-TGr) were induced in CHO cells. The mutation frequency in the Na+/K+-ATPase locus in CHO cells (1.5 X 10(-6) mutants/microM) is higher than that in C3H10T1/2 cells (1.0 X 10(-6) mutants/microM). The maximal mutation frequency at the Na+/K+-ATPase gene locus was attained with 30 min of exposure in C3H10T1/2 cells, whereas the mutation frequency in CHO cells continued to increase up to 80 min of treatment. Similarly, the maximal mutation frequency at the HPRT locus also continued to increase up to 80 min of treatment. Finally, a linear plot of alkali-labile lesions versus 6-TGr mutations was obtained; but the same relationship was not observed in the case of ouar mutation.     

Indirect mutagen assay in human lymphocyte in the presence of a metabolic activation system

Chromosome aberrations in cultured human lymphocytes were examined after exposures to various concentrations (from 1 X 10(-6) to 1 X 10(-3) mol X l-1) of cyclophosphamide (CP) in the presence or absence of a metabolic activation system (S9 mix). With metabolic activation, increases in the frequency of aberrant cells (AB. C.) produced by CP were significant and dose-dependent. At a concentration of 5 X 10(-4) mol X l-1, activated CP induced 29% AB. C. versus 6% AB. C. detected after exposures to CP without metabolic activation. The freshly prepared S9 mix did not virtually differ in its activation potency from the S9 mix stored for 3 weeks at -20 degrees C. CP preincubated for 100 min with S9 mix caused little or no increase in AB. C. frequency above the control level.     

Molecular dosimetry of the mutagen ethyl methanesulfonate in Drosophila melanogaster spermatozoa: linear relation of DNA alkylation per sperm cell (dose) to sex-linked recessive lethals.

The dosage-response curve for EMS was determined with dose measured as ethylations of DNA per sperm cell, and response measured as the relative frequency of sex-linked recessive lethals induced in sperm cells of Drosophila melanogaster. Dose can be converted to ethylations per nucleotide of DNA by dividing ethylations of DNA per sperm cell by 3 X 10(8) nucleotides per sperm cell. Adult males were exposed to equal amounts of either [3H]EMS for determining dose or nonlabeled EMS for determining mutational response. By feeding EMS for 24 h in a concentration of 25 mM, a high dose of 1.4 X 10(-2) ethylations per nucleotide was observed. With 1.4% of the nucleotides ethylated, 57% of the X-chromosomes were hemizygously viable; therefore, ethylation per se is not very efficient in inducing mutations. The relative frequency of mutations increased linearly with the dose from a dose of 2.1 X 10(-4) to 1.4 X 10(-2) ethylations per nucleotide. No threshold was apparent, and the statistical limits of the exponent, 1.0 +/- 0.1, excluded an exponent as high as 1.2. This linear relation suggests no change in mechanism of mutagenesis occurs from low to high dose in Drosophila. A nonlinear relation was found between exposure and dose; when exposure was increased by a factor of 250 (from 0.1 to 25 mM EMS in the feeding medium) dose was increased by a factor of only 68. By extrapolating down from our lowest dose of 2.1 X 10(-4) ethylations per nucleotide with an observed frequency of 0.55% +/- 0.08% sex-linked recessive lethals, we estimate the doubling dose for sex-linked recessive lethals to be 4 X 10(-5) ethylations per nucleotide.     

Chemical reactivity of the functional groups of insulin. Concentration-dependence studies.

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.     

Hormonal regulation of prolactin release by human prolactinoma cells cultured in serum-free conditions

Thyrotropin-releasing hormone (TRH) stimulates the prolactin (PRL) release from normal lactotrophs or tumoral cell line GH3. This effect is not observed in many patients with PRL-secreting tumors. We examined in vitro the PRL response to TRH on cultured human PRL-secreting tumor cells (n = 10) maintained on an extracellular matrix in a minimum medium (DME + insulin, transferrin, selenium). Addition of 10(-8) M TRH to 4 X 10(4) cells produced either no stimulation of PRL release (n = 6) or a mild PRL rise of 32 +/- (SE) 11% (n = 4) when measured 1, 2 and 24 h after TRH addition. When tumor cells were preincubated for 24 h with 5 X 10(-11) M bromocriptine, a 47 +/- 4% inhibition of PRL release was obtained. When TRH (10(-8) M) was added, 24 h after bromocriptine, it produced a 85 +/- 25% increase of PRL release (n = 8). This stimulation of PRL release was evident when measured 1 h after TRH addition and persisted for 48 h. The half maximal stimulatory effect of TRH was 2 X 10(-10) M and the maximal effect was achieved at 10(-9) M TRH. When tumor cells were pretreated with various concentrations of triiodothyronine (T3), the PRL release was inhibited by 50% with 5 X 10(-11) M T3 and by 80% with 10(-9) M T3. Successive addition of TRH (10(-8) M) was unable to stimulate PRL release at any concentration of T3. The addition of 10(-8) M estradiol for up to 16 days either stimulated or had no effect upon the PRL basal release according to the cases. In all cases tested (n = 4), preincubation of the tumor cells with estradiol (10(-8) M) modified the inhibition of PRL release induced by bromocriptine with a half-inhibitory concentration displaced from 3 X 10(-11) M (control) to 3 X 10(-10) M (estradiol). These data demonstrate that the absence of TRH effect observed in some human prolactinomas is not linked to the absence of TRH receptor in such tumor cells. TRH responsiveness is always restored in the presence of dopamine (DA) at appropriate concentration. This TRH/DA interaction seems specific while not observed under T3 inhibition of PRL. Furthermore, estrogens, while presenting a variable stimulatory effect upon basal PRL, antagonize the dopaminergic inhibition of PRL release.     

Thyroid hormone. Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have examined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5 X 10(-8) M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1 X 10(-7) M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5 X 10(-9) M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K'd1 = (0.82 +/- 0.36) X 10(-10) M and K'd2 = (3.2 +/- 0.60) X 10(-8) M. The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1 X 10(-8) M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5 X 10(-8) M T3 showed a K'd1 = (0.15 +/- 0.10) X 10(-10) M and K'd2 = (3.5 +/- 0.10) X 10(-8) M. We conclude that T3 inhibits the action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

The equivalent conductivity of aqueous solutions of alkali metal salts of a number of ionic polysaccharides

Measurements of the equivalent conductivity of aqueous solutions of alkalimetal salts of a number of ionic polysaccharides at 25 degrees C are reported. The polysaccharides studied are: (1) three carboxymethylcelluloses of various degrees of substitution (Li+, Na+, Cs+ salts) in the concentration range 4 X 10(-4) - 6 X 10(-2) equivalents alkali ion per liter, (2) Polypectate (Li+, Na+, K+, Cs+ salts) in the range 1.5 X 10(-4) - 2 X 10(-2) equivalent alkali ion per liter, and (3) Dextransulfate (Li+, Na+, K+ salts) in the range 3 X 10(-4) - 10(-1) equivalent alkali ion per liter. The results are compared to some earlier data and to a limiting law for conductance of rod-like polyions derived by Manning. It is concluded that although qualitative agreement is obtained between observed data and the limiting law when various polyions of different charge densities are compared at a given concentration, the concentration dependence predicted by the limiting law is in agreement with the observed curves only for polyions of a relatively low charge density. At higher charge densities appreciable deviations occur, and dextransulfate which does not have the rod-like polyion structure required by theory does not conform to the predicted concentration dependence at all.     

Interaction of piracetam with 3H-imipramine binding sites and the GAMA-benzodiazepine receptor complex of brain membranes

Piracetam at a concentration of 10(-6) M was shown to behave as a noncompetitive inhibitor of 3H-imipramine specific binding to rat brain membranes. At the same time piracetam failed to influence specific binding of 3H-mianserin to membranes of guinea-pig cerebellum, which is indicative of its inability to suppress histamine H1 receptors, a component of 3H-imipramine specific binding sites. At a concentration of 10(-4) M piracetam does not change specific binding of 3H-flunitrazepam to rat hippocampal membranes in the absence of GABA, but in the presence of 5 X 10(-5) M GABA, like atypical tranquilizer mebicar, acts as a competitor of 3H-flunitrasepam binding. Though Ro-15 1788 did not suppress anxyolytic piracetam (and mebicar) effect, our results give evidence of a possible involvement of GABA-benzodiazepine supramolecular complex in the anxiolytic activity of piracetam.     

Mitomycin-C-induced sister-chromatid exchanges and cell-cycle kinetics in lymphocytes from patients with Klinefelter syndrome

The chromosomal sensitivity to mitomycin-C (MMC) and cell-cycle kinetics in cells from patients with Klinefelter syndrome, a sex chromosomal disorder giving a high risk of malignant tumor, were studied by techniques of sister-chromatid exchanges (SCEs). The frequencies of MMC-induced SCEs increased in proportion to the increase in MMC concentration in both patient and normal control cells. At low levels of MMC there were no significant differences in SCE frequencies between the patient and normal control cells, but at MMC concentrations of 3 X 10(-8) M (p less than 0.05) and 1 X 10(-7) M (p less than 0.01), significant increases in the frequency of MMC-induced SCEs were observed in cells from patients compared to cells from normal controls. Although the analysis of cell-cycle kinetics both after various culture times and after treatment with MMC revealed that there were no significant differences between the patient and normal control cells, patients with Klinefelter syndrome showed a tendency to cell-cycle delays after treatment with MMC in comparison with normal controls.     

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...     

Characterization of specific high-affinity receptor for human lymphotoxin

The specific cell surface receptors for lymphotoxin (LT) which are expressed on murine fibroblast L.P3 cells, a subline of L929 cells, were found to consist of a single class of specific high-affinity receptors with a dissociation constant (Kd) of 3.8 X 10(-10) M and a density of 5.8 X 10(3) sites/cell. Similarly, murine fibroblast L929 cells, human melanoma A375 cells and human cervical carcinoma HeLa-S3 cells had about 7.2 X 10(3), 3.5 X 10(3), and 6.6 X 10(3) sites/cell with Kd values of 1.4 X 10(-10), 0.5 X 10(-10), and 1.1 X 10(-10) M, respectively. Among the LT receptor-positive cell lines, there was no direct correlation between the level of specific LT binding and the sensitivity to the cytotoxic or cytostatic effect of LT. Cross-linking of 125I-LT to the cell surface receptors with disuccinimidyl suberate, followed by two-dimensional gel electrophoresis of the cell lysate, revealed two kinds of LT-LT receptor complexes with molecular weights of 70 and 97 kDa, and having the same pI value of 6.8. Cell-bound 125I-LT was internalized within 1 h and degraded intracellularly, and finally secreted into the medium within a few hours. Appropriate concentrations of LT and interferon gamma (IFN gamma) showed synergistic cytotoxicity toward murine fibroblast L.P3 cells and human monocytoma U937 cells, but these cytokines were only slightly cytotoxic individually. Preincubation of these cells with IFN gamma increased the total number of LT receptors without any significant change in the dissociation constant or in the molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the adenine level of a culture medium on the frequency of revertants in haploid yeasts

The frequency of reversion from adenine auxotrophy in the yeast Saccharomyces cerevisiae increases with the decrease of adenine concentration in the medium: for the 8PG-59 strain (a ade2-192 rad2) the reversion frequency is 1.7 X 10(-8), 3.2 X 10(-6) and 1.8 X 10(-5) per cell division, the initial adenine concentrations being 10, 1 and 0.1 mg/l, respectively. An increase of the reversion frequency with the culture age in the stationary phase of growth is demonstrated using an improved method of registration of revertants, with the initial concentration of 0.1 mg/l of adenine in the medium. The reversion frequency was 9.1 X 10(-7) on the 7th day, 1.8 X 10(-6) on the 10th day and 1.8 X 10(-5) on the 14th day.     

The porcine ovarian luteinizing hormone/human chorionic gonadotropin receptor II. Is the purified receptor an oligomer of identical subunits?

Purified porcine luteinizing hormone/human chorionic gonadotropin receptors were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis following reduction and thermal denaturation and stained with Coomassie Brilliant Blue. A major protein of Mr = 77 +/- 4 X 10(3) and a minor protein of Mr = 66 +/- 4 X 10(3) were observed. Iodoreceptor proteins were resolved into a major component of Mr = 77 +/- 3 X 10(3) and a minor component of Mr = 62 +/- 5 X 10(3) after reduction and thermal denaturation. In the absence of reduction, the iodoreceptor had a major component of Mr 63 +/- 3 X 10(3). Purified human chorionic gonadotropin specifically transferred part of the iodoreceptor from the Mr = 63 X 10(3) species to an Mr = 110-120 X 10(3) species. Purified receptors were analyzed by nondenaturing polyacrylamide gel electrophoresis and identified by specific binding of iodo-human chorionic gonadotropin. Three binding species with approximate Mr = 60 X 10(3), 130 X 10(3), and 260 X 10(3) were identified. Iodoreceptors co-migrated with the Mr = 60 X 10(3) species under the same conditions. Similar results were obtained following renaturation of receptors separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reduction and thermal denaturation. These results suggest for the first time that the porcine corpus luteum luteinizing hormone/human chorionic gonadotropin receptor may be a hormone binding monomer of Mr = 60-65 X 10(3), and that the monomer may associate to form hormone binding polymeric receptor complexes.     

Stronger affinity of reticulocyte release factor than natural suppressor tRNASer for the opal termination codon

Animal natural suppressor tRNA did not affect the release reaction of reticulocyte release factor (RF) at the same concentration of tRNA (both estimated as being present at a similar level of 3-5 X 10(-8) M in vivo); even at a 10-fold greater concentration the tRNA did not prevent the release reaction with RF. In order to confirm this result, the Ka values were determined. The Ka value between RF and UGA was 1.26 X 10(6) M-1 and that between the suppressor tRNA and UGA amounted to 8 X 10(3) M-1. This result showed that RF had a 150-fold stronger affinity than suppressor tRNA for the opal termination codon. Incorporation of phosphoserine into phosphoprotein via phosphoseryl-tRNA was inhibited by addition of RF to the reaction mixture. These results suggest that animal natural suppressor tRNA in the normal state does not perform its suppressor function, except in special cases where mRNA has the context structure near the opal termination codon (UGA).