Yoshiaki Maeda

Efficient transformation of Mesorhizobium huakuii subsp. rengei and Rhizobium species.

The transformation of Mesorhizobium huakuii subsp. rengei B3 with either pBBR122 or pKT230 was carried out. We determined the optimal conditions required for transformation by electroporation and obtained up to 10(5) CFU/microg pBBR122. Plasmids prepared from strain B3 yielded higher transformation efficiency than those from various dam or dcm mutant strains of Escherichia coli. This result suggests that a high transformation efficiency is not related to the methylation of plasmid DNA in E. coli. Using the optimal conditions for electroporation, we performed transformation of several species of Rhizobium, Mesorhizobium and Sinorhizobium. All tested strains of these species were transformed with pBBR122. Strains of M. huakuii and R. phaseoli AHU1133 were transformed at high efficiency, whereas transformation efficiencies of Rhizobium sp. NGR234 and S. meliloti strains were less than 2 x 10(3) CFU/microg plasmid DNA.

Detection of estrogen receptor-like high affinity components by exposure of cytosols from mouse leydig cell tumor or male rat liver to chaotropic salts☆

Abstract Modifications of steroid-macromolecule interaction induced by so-called chaotropic salts were studied. When the cytosol from one of estrogen-independent mouse Leydig cell tumors was incubated with [3H]-estradiol (E2), an unusual estrogen binding component was identified, with a relatively weak affinity for E2 (Kd 10−7 M). This binding could not be inhibited by diethylstilbestrol (DES). However, exposure of this cytosol to 0.4 M NaSCN resulted in the clear demonstration of high affinity binder for E2 (Kd 10−9 M). Furthermore, this high affinity binding with E2 was inhibited by DES in a competitive manner. The quantitation of the binding sites revealed that the binding capacity at low concentrations of [3H]-E2 (less than 10 nM) is higher in the presence of NaSCN than in the absence of NaSCN. Liver cytosol from noncastrated male rat was also found to contain E2 binding component with a low affinity (Kd 10−7 M) and a very high concentration of sites. The binding component with high affinity (Kd 10−9 M) as well as a concomitant loss of the low affinity sites became detectable in 0.4 M NaSCN. Moreover, this association with E2 was found to be competitively inhibited by DES (Ki 3 × 10−9 M). The similar changes were elicited by the other chaotropic salt, NaClO4. These observations suggest that the detection of receptor-like high affinity binder in chaotropic salts may be mediated by two different mechanisms: one is a formation of new high affinity binder, the other is a destruction of the unusual second binder. Furthermore, the easy demonstration of estrogen receptor in chaotropic salt which is not detectable in a conventional buffer system might indicate that careful consideration is required to decide the absence of receptor protein.

STEROID RECEPTOR FORMS AND THEIR INTERACTION WITH CYTOPLASMIC MODULATORS

The cytoplasmic modulator affecting steroid receptor functions was studied. The diminution in the concentration of the low molecular weight substances in the cytosol caused the increased interaction of hormone-receptor complexes with nuclei (a step termed activation). Furthermore, dialysis of rat uterine cytosol in the absence of estrogens subsequently followed by incubation with isolated nuclei resulted in the demonstration of an appearance of unoccupied nuclear receptor which was found to be 4S form. The addition of the dialyzable compound into rat uterine estrogen receptor system caused the suppression of temperature-dependent activation while the already-activated estrogen receptor was not affected by the small molecules in relation to its nuclear binding ability. These results may indicate that this small molecule, so-called the low molecular weight inhibitor, is capable of interacting with nonactivated receptor. In one of the estrogen-independent Leydig cell tumor lines, unique low-affinity estrogen binder with a mol. wt approximately 36,000 was identified. This binder did not show appreciable nuclear binding ability even after conventional heat activation. However, removal of the small molecules from this cytosol resulted in a marked increase in affinity for estrogens with a concomittant alteration of the mol. wt to approximately 70,000. These changes also paralleled a dramatic enhancement of nuclear binding ability. This small molecule might be different from the low molecular weight inhibitor suppressing receptor activation, since this tumor cytosol containing unique estrogen binder had much less inhibitory activity against receptor activation when compared with those in the other cytosol containing usual estrogen receptor systems. In adult rat urine cytosol, a new modulator was identified to recognize only activated estrogen receptor but not glucocorticoid receptor and to induce receptor aggregation with a concomittant loss of its nuclear binding ability. These reactions were accelerated by the presence of physiological or higher KCl concentration and exposure to a relatively high temperature (20-30 degrees C). Interestingly, this factor was not identified in the immature rat uteri or liver.