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To examine the role of Ia molecules in T cell responses to allo-class I major histocompatibility antigens, a series of allo-class I-reactive T cell hybridomas was established. Of 134 T cell hybridomas obtained from the fusion of C3H/HeJm or B10.HTT T cells stimulated with C57BL/6 splenocytes, nine T cell hybridomas were reactive to class I antigens and 126 T cell hybridomas were reactive to class II antigens. Six of the nine IL 2-producing T cell hybridomas were further analyzed: five mapped to H-2Kb and the other mapped to H-2Db. Three of these T cell hybridomas, HTB-157.7, HTB-176.10, and HTB-177.2, could react to the EL-4 cell line that expresses H-2Kb and H-2Db class I antigens but lacks class II I-Ab molecules. Furthermore, the activation of these three T cell hybridomas with C57BL/6-derived splenocytes was not blocked by either anti-I-A or anti-L3T4 antibody. In contrast, the other three T cell hybridomas, CB-127.6, CB-221.7, and HTB-102.7, failed to react with EL-4 but reacted with the LB cell line which expresses class I (H-2Kb, H-2Db) and class II (I-Ab) molecules. Although class II molecules were required for activation of the latter clones, there was no apparent I-A allele specificity, suggesting that a relatively nonpolymorphic Ia determinant was involved. The activation of the three latter T cell hybridoma clones with C57BL/6 splenocytes could be blocked completely by either anti-I-A or anti-L3T4 antibody. The data are interpreted in terms of possible T cell receptor models for recognition of class I with nonpolymorphic class II determinants.

Studies outlined here compare the properties of mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in cytosol from adrenalectomized mouse brain. Pretreating cytosol with dextran-coated charcoal (DCC) produced a 4.7-fold increase in the subsequent macromolecular binding of the mineralocorticoid, [3H]aldosterone (20 nM ALDO, in the presence of a 50-fold molar excess of the highly specific synthetic glucocorticoid, RU 26988), whereas it produced a 55% decrease in the binding of the glucocorticoid, [3H]triamcinolone acetonide (20 nM TA). Scatchard analyses revealed that DCC pretreatment had no effect on the affinity or maximal binding of Type I receptors for [3H]ALDO (in the presence of a 0-, 50- or 500-fold excess of RU 26988), whereas it produced a 3- to 6-fold increase in the Kd, and an 8-43% decrease in the maximal binding, of Type II receptors for [3H]TA and [3H]dexamethasone. Optimal stability of unoccupied Type I receptors at 0 degree C was found to be achieved in buffers containing glycerol, but lacking molybdate. Although the addition of molybdate was found to reduce the loss in Type I receptor binding observed after incubating unlabelled cytosol at 12 or 22 degrees C, this stabilization was accompanied by a concentration-dependent reduction in the binding of [3H]ALDO at 0 degree C. Scatchard analyses showed that this reduction was due to a shift in the maximal binding, and not the affinity, of the Type I receptors for [3H]ALDO. The presence or absence of dithiothreitol in cytosol appeared to have little effect on the stability of Type I receptors. In contrast to our finding for Type I receptors, it was possible to stabilize the binding capacity of unoccupied Type II receptors, even after 2-4 h at 12 or 22 degrees C, if the glycerol containing buffers were supplemented with both molybdate and dithiothreitol. In summary, these results indicate distinct chemical differences between Type I and Type II receptors for adrenal steroids.

In order to investigate whether inhibin (FSH-suppressing activity) is present in human follicular fluid (hFF) and whether inhibin in hFF could be correlated with the FSH level in peripheral serum, the effect of hFF on FSH secretion was studied using monolayer culture of rat anterior pituitary cells. Dextran-coated charcoal (DCC)-treated hFF exerted an inhibitory effect on pituitary FSH secretion but not on LH. The inhibitory effect of hFF upon basal FSH secretion was different from those of steroids such as estradiol, progesterone, testosterone and androstenedione. hFF inhibited the LHRH-stimulated secretion of both FSH and LH. Since hFF and porcine follicular fluid (pFF) produced a parallel dose-dependent decrease of basal FSH secretion, the same suppressing activity may be present in both hFF and pFF. Inhibin activity increased gradually during the follicular phase, but decreased in the luteal phase. Inhibin activity in hFF except during preovulatory surge showed a significant inverse correlation with the FSH level in peripheral serum. These results might indicate that inhibin activity in hFF increased according to follicular maturation and that inhibin (non-steroidal substance) may be one of the important regulators of FSH secretion in the human pituitary.

No abstract available.

Estrogen receptor (ER) in the C3H mouse mammary tumor was characterized in the cytosol and determined in the nuclear KCl extract and the nuclear KCl unextractable fraction (non-extract). In the cytosol, the estradiol (E2)-ER complex was sedimented in the 8S region by 5-20% linear sucrose gradient centrifugation. A steroid specificity study showed the presence of estrogen-specific binder with a dissociation constant (Kd) of approximately 2 nM. ER levels in the cytosol, the nuclear KCl extract, and the suspension of nuclear KCl unextractable fraction were determined by Scatchard analyses, using the dextran-coated charcoal (DCC) adsorption method for the former two and the sedimentation method for the latter. Although ER was present in the nuclear fractions, it seems more plausible that cytosol ER contributes to nuclear translocation of the estrogen-ER complex. The results suggest the presence of an intranuclear mechanism for the estrogen action.

Clinical and experimental evidence suggests a role for estrogen in the natural history of desmoid tumors (DT). Antiestrogen (tamoxifen) has been used empirically in some patients with significant tumor regression. To further investigate the mechanism of hormonal influence on desmoid tumors we initially characterized the cytosol estrogen receptor (ER) and antiestrogen binding sites (AEBS) in microsomal fractions of 15 cases of DT. Biopsy specimens were obtained from nine female and six male patients. ER assay was determined in cytosol (105,000 g) and the AEBS was detected in the microsomal fraction (7000 g for 20 min) by a DCC assay technique. ER was present in 33% of DT assayed (5/15), with equal incidence in males and females. Receptor content in female patients was higher than in male patients (26.52 +/- 16 vs 10.82 +/- 8.32 fmol/mg protein). Dissociation constant (Kd) range (0.44-3.97 nM) was well within the values seen in other estrogen target tissues. The AEBS were detected in 79% of the cases. The mean binding value was 236.7 +/- 170.2 fmol/mg protein. Kd values were between 0.39 and 5.97 nM. ER settled predominantly in the 4S region and AEBS settled in the 5-5.5S region in a 5-20% sucrose gradient. AEBS was detected in seven patients with negative ER. No correlation between ER and AEBS contents was observed. Competition studies revealed minimal binding with either DEX, DHT, R5020, and R1881, but partial binding with tamoxifen in cytosol and estradiol in microsomal fractions. ER and AEBS assays may be of prognostic significance in the natural history of these tumors.

The rate of uptake of radioactive phosphatidylcholine molecules of different fatty acid composition in intact erythrocytes as facilitated by a phosphatidylcholine-specific transfer protein has been studied. When trace amounts of radiolabeled phosphatidylcholine molecules are present in donor vesicles consisting of egg phosphatidylcholine and cholesterol, the transfer of the radiolabeled species depends strongly on their fatty acyl composition: dipalmitoylphosphatidylcholine is transferred at the lowest rate, 1-saturated-2-unsaturated species are transferred faster and the highest rate is observed for dioleoyl phosphatidylcholine. Transfer of the various phosphatidylcholine molecules was measured furthermore using donor systems in which the bulk phosphatidylcholine was varied in its fatty acyl composition. Also in this type of experiment, the transfer protein preferentially stimulated transfer of unsaturated phosphatidylcholine molecules, especially from an environment containing more saturated molecules. Finally, the efflux of labeled phosphatidylcholine from intact erythrocytes to plasma in the absence of the phosphatidylcholine-specific transfer protein was studied and it became clear that in this case the nature of the effused molecules itself, rather than the composition of the bulk lipids, determined the effuse rates. An important conclusion to be drawn from these experiments is that radiolabeled phosphatidylcholine molecules, when used as markers for phospholipid exchange or transfer, should resemble in their fatty acid composition the composition of the bulk lipid in order to provide reliable data on rates and extents of the process studied.