Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

Mg2+-, Ca2+-dependent unwinding of DNA by poly-L-glutamic acid

In order to examine a possibility that the high acidic amino acid region in the nonhistone protein HMG(1+2) is concerned with the Mg2+-, or Ca2+-dependent unwinding of DNA by the HMG(1+2) (1,2), poly-L-glutamic acid was employed as an acidic model peptide for thermal melting temperature analysis. The poly-L-glutamic acid bound to DNA either in the presence or absence of Mg2+. The poly-L-glutamic acid unwound DNA double-helix to a similar extent to HMG(1+2) in the presence of Mg2+ or Ca2+, but not in the absence of them. These results may suggest that the high acidic amino acid region in HMG(1+2) participates in Mg2+-, Ca2+-dependent unwinding of DNA double-helix.     

Effect of thyrotropin on glycosaminoglycans synthesized by primocultured thyroid cells

The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin. After labelling with [³H] glucosamine and [³⁵S] $\text{SO}{}_4{}^{\mathrm{2?}}$, enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations. They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-β-galactosidase. Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid [³H] label and, when cells organized into follicles, of the proportion of cell-associated [³H] GAGs. This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates.     

Accurate assay of dopa decarboxylase by preventing nonenzymatic decarboxylation of dopa

The nonenzymatic decarboxylation of dopa was completely blocked by both 2-mercaptoethanol and EDTA together over the wide range of pH. This finding made it possible to measure the activity of dopa decarboxylase precisely even at an alkaline pH value. The pH optimum of dopa decarboxylase was found to be pH 7.0 and the Km value for dopa was determined to be 4 X 10(-5) M.     

Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection

A new method for the determination of tryptophan and its metabolites in a single mouse brain using high-performance liquid chromatography (HPLC) with fluorometric detection is described. Tryptophan, serotonin, 5-hydroxyindoleacetic acid, indoleacetic acid, and tryptophol were clearly separated by a C8 reverse-phase column. Tissue preparation is performed only to centrifuge homogenates of brain prior to the injection to HPLC. The sensitivity is in the range from 10 to 15 pg.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

The sphingomyelin pools in the outer and inner layer of the human erythrocyte membrane are composed of different molecular species

Analyses of the fatty acid composition of the outer and inner pools of sphingomyelin in the human erythrocyte membrane revealed significant differences in molecular species composition of these two pools. The sphingomyelin in the inner monolayer, representing 15–20% of the total sphingomyelin content of this membrane, is characterized by a relatively high content (73%) of fatty acids, which have less than 20 carbon atoms, whereas these account for only 31% of the total fatty acids in the sphingomyelin in the outer leaflet. On the other hand, the ratio saturated/unsaturated fatty acids in the two pools is similar. Significant differences are also observed for the fatty acid composition of the sphingomyelin in human serum when compared to that in the outer monolayer of the corresponding red cell. These results are interpreted to indicate an (almost) complete absence of transbilayer movements of sphingomyelin molecules in the human erythrocyte membrane, whereas an exchange of this phospholipid between the red cell membrane and serum is either virtually absent, or affects only a minor fraction of the sphingomyelin in the outer membrane layer.     

The determination of the separate Ca pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47–72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.     

Microbial fermentative preparation of L-[15N2]lysine and its tracer: application to serum amino acid kinetic studies

The microorganism Brevibacterium flavum 21129 has been used to produce multigram batches of L-[15N2]lysine of high purity and isotopic enrichment by supplementation of the growth medium with (15NH4)2SO4 of 98.0 atom% excess. The doubly 15N-labeled lysine can be detected at dilutions 10 times greater than singly labeled lysine when isotope dilution curves are analyzed by gas chromatography-mass spectrometry. This enhanced sensitivity permits kinetic measurements of plasma free-lysine isotope content over a 300-fold dilution during 6 h following a single oral bolus of 5 mg/kg body wt. This inexpensive preparation method lends itself to the production of highly useful biochemical compounds for kinetic studies of human nutrition.