An estrogen receptor from xenopus laevis liver possibly connected with vitellogenin synthesis

This paper describes an estrogen receptor which is found in both the nucleus and cytoplasm of liver cells from male Xenopus laevis, and which seems to be involved in the induction of vitellogenin synthesis. It has a high affinity for estradiol (K_d = 0.5 × 10^?9 M), and the affinities of various steroids for the receptor correlate well with their ability to induce vitellogenin synthesis. It sediments at 3.5S at 0°C in 0.5 M KCI. The rate of sedimentation is unaffected by incubation at 20°C prior to centrifugation, but increases if the salt concentration is lowered to 0.1 M KCI or to zero. It has a Stokes radius of 2.6 nm and a molecular weight of approximately 40, 000. The receptor is present at very low levels compared to other steroid target tissues (50–100 fold less than chick oviduct). The cytoplasm of a single hepatocyte contains 92 ± 18 binding sites for estradiol, while each nucleus contains 99 ± 19 sites.     

2-Aminoethylphosphonic acid concentrations in some rumen ciliate protozoa

The concentration of 2-aminoethylphosphonic acid has been measured in seven genera of rumen ciliate protozoa. Expressed as milligrams per gram of total nitrogen, 2-aminoethylphosphonic acid concentrations ranged from 17.2 in Ophryoscolex spp. to 72.4 in Eremoplastron spp.     

Effects of Monovalent and Divalent Cations on 3-(+)[125I]Iododizocilpine Binding to the N-Methyl-d-Aspartate Receptor of Rat Brain Membranes

We have investigated the binding of 3-[125I]iododizocilpine ([125I]iodo-MK-801) to the N-methyl-D-aspartate (NMDA) receptor in well-washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine greater than thienylphencyclidine greater than phencyclidine greater than ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS-19755, 7-chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 microM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.     

Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low-Km GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with less than 10% of the sites in the high-affinity state. Both sites were also detected at 4 degrees C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the Bmax for [3H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic degradation of heterologous proteins expressed inAspergillus niger

Summary Proteolytic degradation of heterologous proteins expressed in the filamentous fungusAspergillus niger reduces the yield of authentic target protein. The activities ofA. niger proteases are differentiated by their effects on two proteins expressed and secreted fromA. niger: hen egg-white lysozyme and porcine pancreatic phospholipase A₂.     

Promoter methylation and progressive transgene inactivation inArabidopsis

Agrobacterium-transformedArabidopsis plants were generated and the stability of their T-DNA-encoded resistance to kanamycin was examined. Of seven families, each homozygous for a single insertion event, two showed progressive inactivation of resistance over four generations of inbreeding. Loss of resistance was associated with methylation of anSst II site in thenos promoter of the kanamycin resistance gene. Treatment of plant roots from inactive lines with the demethylating agent 5-azacytidine restored the ability of such lines to form callus on kanamycin-containing media. These observations are consistent with the view that methylation is a factor in the progressive inactivation of transgenes inArabidopsis.