Influence of androgen on the development of sexual behavior in the rat. II. Time and dosage of androgen administration during the neonatal period and masculine and feminine copulatory behavior in females

The objective of the present study was to delineate the period of sensitivity to a single androgen exposure during the initial neonatal hours on the development of masculine and feminine copulatory behavior in female rats. Female rats were injected once with either 500, 50, or 5 micrograms testosterone propionate (TP) at either 1 or 24 hr after birth. Following castration in adulthood and TP replacement, the females were tested four times at weekly intervals in prolonged sessions for masculine copulatory behavior. One month following the masculine copulatory tests the females were tested for 3 weeks for feminine copulatory behavior with weekly increasing levels of estradiol benzoate (2.5, 10, and 25 micrograms) and progesterone (200 micrograms). The results demonstrate that a single injection of TP administered at either 1 or 24 hr after birth can significantly increase the capacity of female rats to exhibit ejaculation patterns and that the amount of androgen that is administered is critical in determining the levels of ejaculatory responding. Similarly, the females given high doses (50 and 500 micrograms) of TP at either 1 or 24 hr neonatally were almost completely defeminized. In contrast, however, the females treated with 5 micrograms TP at 1 and 24 hr showed different levels of lordotic performance indicating a greater sensitivity to androgen immediately after birth than at 24 hr in female rats as has been shown in male rats.     

Proliferation of chicken peripheral blood leukocytes in response to pokeweed mitogen is macrophage dependent

Chicken peripheral blood leukocytes (PBL) proliferate in vitro in response to a wide range of pokeweed mitogen (PWM) concentrations. The PWM response was not influenced by the major histocompatibility complex (MHC) haplotype nor by the intrinsic responsiveness of the PBL to concanavalin A (Con A). The results indicate that PWM under our conditions stimulates B-lineage cells, although a T-cell subset is also clearly induced to division. The PBL from B-cell-depleted animals gave substantially lower responses than those from normal controls. Pokeweed mitogen stimulation of PBL was adherent cell dependent. Thus the low PWM response of adherent cell-depleted PBL was reconstituted by the addition of irradiated unseparated PBL. Furthermore, purified irradiated adherent cells containing greater than 90% macrophages were also able to reconstitute PWM responses. Finally, we have shown that PWM was able to induce large numbers of B cells to produce cytoplasmic immunoglobulin. However, only a minor proportion of such cells were induced to immunoglobulin secretion.     

Retention deficit for avoidance training in hypophysectomized rats: Time-dependent enhancement of retention performance with post-training ACTH injections

These experiments examined the effects of hypophysectomy on retention of avoidance training. In addition, the experiments examined the effects, on retention, of post-training ACTH injections administered to hypophysectomized rats. Rats were trained in a visual discriminated avoidance Y maze. Each rat received six training trials followed by six retraining trials the next day. Retention was measured by the number of correct choices during the retraining trials. When trained with a low-footshock intensity (0.8 mA), hypophysectomized rats showed retention performance which was significantly poorer than that of intact animals. There was no significant difference in performance when the animals were trained with a higher footshock intensity (1.4 mA), in part because of poorer retention performance of intact animals under these training conditions. Under both footshock conditions, a single post-training injection of ACTH enhanced later retention performance of hypophysectomized rats. This effect on memory was timedependent; injections delayed 2 or 6 hr after training did not significantly enhance retention. These findings are consistent with the view that hormonal responses to training may modulate later retention of the training experience.     

The estrogenic arousal of aggressive behavior in female mice

Experiments were conducted to determine the conditions under which estrogen would promote male-like aggressive behavior in female mice. The results of the first experiment showed that most females chronically exposed to testosterone propionate (TP) in adulthood fought, whereas females similarly treated with estradiol benzoate (EB) did not display aggression. Another experiment found that, when either TP or EB was administered on the day of birth, adult females displayed aggression in response to daily EB injections during adult life. Also, the potentiating effect of neonatal hormone exposure declined over the first 12 days postpartum, as 100% of the Day 0, 75% of the Day 6, and 0% of the Day 12 and 18 TP-treated females fought in response to daily injections of 40 μg of EB in adulthood. The final study showed that, under the test conditions employed, the failure of a chronic adult EB regimen to promote aggression was not due to a competing tendency to display female sexual behavior.     

Reactivity of the B6.C-H-2bm−1 mutant to the wild-type tumor EL4: Characterization of the serological response

Hyperimmunization of B6.C-H-2^bm?1 (H-2^bm?1), a congenic mutant of C57Bl/6J (B6), with the C57Bl lymphoma EL4 resulted in the induction of antibodies with apparent EL4 specificity. EL4 reactivity was demonstrable in H-2^bm?1 anti-EL4 sera by complement-mediated cytotoxicity, absorption, and enzyme-linked immunosorbent assay. By these same serological tests, H-2^bm?1 anti-EL4 serum was found to be nonreactive with B6 normal lymphoid cells, embryonic fibroblasts, and two fibrosarcomas previously induced in B6 mice by methylcholanthrene. These data suggest that the serological response of H-2^bm?1 to EL4 is directed against tumor-associated antigens on EL4. These findings indicate that congenic mutants which differ from the wild-type strain at MHC Class I subloci, but which do not evoke serological responses to MHC components, may provide convenient sources for preparing serological reagents directed against tumor-specific antigens.     

Non-equivalence of the carboxyl groups of glucagon in the carbodiimide-promoted reaction with nucleophiles and the role of carboxyl groups in the ability of glucagon to stimulate the adenyl cyclase of rat liver

The polypeptide hormone glucagon can react with the nucleophiles; glycinamide, taurine or ethylenediamine in the presence of 1-ethyl-3-(3-dimethylaminopropylcarbodiimide). The number of carboxyl groups which are modified depend on the concentration of guanidine hydrochloride in the reaction media. These results demonstrate an additional property which glucagon possesses in common with larger globular proteins and suggests that the hormone has a specific, folded structure in dilute aqueous solution. In the absence of guanidine hydrochloride only one taurine residue is incorporated into the terminal carboxyl group of the peptide. In 7 M guanidine hydrochloride all four of the carboxyl groups react with glycinamide or taurine while only two and a half residues of ethylenediamine are incorporated. All of these derivatives and glucagon have identical circular dichroism spectra in dilute aqueous solution. The taurine modified derivative has greatly enhanced solubility compared with glucagon but still associates to structures of higher helical content. Both of the taurine derivatives of glucagon have the ability to stimulate the adenyl cyclase of rat liver membranes but at concentrations several fold higher than is needed for the native hormone. It is suggested that each carboxyl group contributes to the binding of the hormone to the specific membrane receptor sites.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Feedback suppression of the immune response in vivo. I. Immune B cells induce antigen-specific suppressor T cells

Stimulated lymphocytes are capable of synthesizing and secreting a variety of lymphokines which can affect the functions of several types of target cells. We report here the existence of a soluble factor released by activated human mononuclear leukocytes which produces a selective inhibition of human pulmonary fibroblast migration. This fibroblast migration inhibitory factor (FIF) was produced by antigen- or mitogen-stimulated human peripheral blood mononuclear leukocytes (PBML) and purified T cells. It inhibited the migration of ⁵¹Cr-labeled fibroblasts in a dose-dependent fashion with optimal effect (65–70% inhibition) obtained at 1:10 dilution and 8–20 hr of incubation. Sephadex G-100 fractionation revealed most activity to be found between 28,000 and 34,000 daltons. FIF was stable at 56 °C for 15 min, but destroyed at 80 °C or at low pH. This factor may play an important role in the modulation of fibrogenesis and healing processes by the immune system.     

Function of rabbit kidneys in vitro at normothermia following equilibration with 3.0 M Me2SO and removal by hypertonic washout at 10 degrees C

In the present study, rabbit kidneys were assayed for function on a 37 °C in vitro perfusion system after perfusion on a 10 °C perfusion system which permits the slow introduction and removal of cryoprotectant. The final concentration of 3.0 M Me₂SO was introduced slowly at two different rates. The washout was achieved by perfusion with Me₂SO-free solutions made hypertonic with mannitol. Two regimens of washout were used: 800, 700, 600, 500, and 400 mOsm/kg; and 600, 500, and 400 mOsm/kg.During perfusion at 37 °C, the glomerular filtration rate was similar in all groups and this increased significantly in all groups with time. Protein leakage was minimal. All three Me₂SO groups showed a depressed Na reabsorption capacity, but the 800 mOsm group was the most severely affected. This was also found with glucose reabsorption. We concluded that rabbit kidneys will function well with the cryoprotectant Me₂SO up to 3 M concentration when introduced slowly and washed out with hypertonic mannitol beginning at 600 mOsm/kg. When 800 mOsm is used at the initial step, the proximal tubular function is severely affected.     

The effect of proteases on endothelial cell migration in vitro

This communication reports on the role of proteases in the migration of endothelial cells in vitro. Endothelial cell (EC) migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Treatment with mitomycin C inhibited wound-induced proliferation of endothelial cells without affecting migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro in 10% serum was not affected by depletion of plasminogen, which inhibited plasmin production, or by various protease inhibitors: soybean trypsin inhibitor, Trasylol, E-amino caproic acid (EACA), ovalbumin, p-tpsyl-1-arginine-methyl ester (TAME), and benzamidine. However, migration and proliferation of endothelial cells in vitro was inhibited by acid-treated serum, a procedure commonly used to inactivate protease inhibitors. Migration of bovine smooth muscle cells, 3T3 cells and SV40-3T3 cells was inhibited by plasminogen-depleted serum; reconstitution with purified plasminogen reversed the depressed migration of only SV40-3T3. These results indicated that endothelial cell migration in vitro is not dependent on plasminogen, which may be another unique property of endothelial cells.     

Butanedione treatment reduces receptor binding of a lysosomal enzyme to cells and membranes

Treatment of the lysosomal enzyme, α-L-iduronidase, with 2,3 butanedione, an arginine modifying reagent, under conditions where enzyme activity was unaffected, reduced by 50% the internalization of the enzyme into cultured human fibroblasts. The lowered rate of internalization was a result of a reduced binding of the enzyme to cell surface receptors. The butanedione treatment of α-L-iduronidase caused a 90% reduction of binding when isolated fibroblast membranes were used as the source of receptor. This marked reduction of binding was also seen when membranes from a rat chondrosarcoma were examined. Although there is ample evidence that the receptor recognizes mannose 6-phosphate residues on the enzyme, the results suggest that other structural features, such as arginine moieties, may also be important in iduronidase binding.     

Prostaglandin synthesis by lymphoid tissue of mice experiencing a graft-versus-host reaction: relationship to immunosuppression

Studies were carried out on the induction of PGE synthesis during the GVH reaction and its role in GVH-induced immunosuppression. The results demonstrated that spleen, lymph node cells and, to a much lesser degree, thymus cells obtained from adult C57BL/6 × AF₁ mice treated with 50–75 × 10⁶ C57BL/6 lymphoid cells were stimulated to produce PGE during the course of the GVH reaction. The spleen and lymph node PGE production peaked at Day 9 post-GVH induction (30- and 15-fold higher than normal, respectively). Thereafter, it declined to near normal levels by Days 25–30 post-GVH induction. Passage of GVH spleen cells through a rayon column removed macrophages but not mitogen-responsive T and B cells and also removed nearly all of the PGE-producing cells, except during the later course of the GVH reaction. Removal of PGE-producing cells from GVH-immunosuppressed spleen cells significantly reconstituted the mitogen response to PHA and LPS. Treatment of mice experiencing a GVH reaction with indomethacin delayed the onset of suppression of the plaque-forming cell response to sheep erythrocytes. These results suggest that early GVH-induced immunosuppression which may represent an amplified normal regulatory mechanism is mediated by increased macrophage production of PGE which suppresses both B- and T-cell functions, whereas at later stages other immunosuppressive mechanisms become operational.     

Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity

The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.     

Modification of the C-terminal octapeptide of cholecystokinin with a high-specific-activity iodinated imidoester: preparation, characterization, and binding to isolated pancreatic acinar cells

Proteoglycans were separated by high-performance liquid chromatography (HPLC), using two coupled Aquapore columns containing glycerylpropylsilane groups covalently linked to large-pore (50–100 nm) silica spheres. This two-column HPLC system was effective in separating cartilage proteoglycan aggregates and monomers, without altering their biochemical integrity. This system was also effective in resolving small amounts of isotopically labeled proteoglycans synthesized by cultured mammalian cells. The small sample size, short analysis time, and high reproducibility represent improvements in the study of proteoglycans over conventional soft-gel chromatography.     

The suppression of experimental allergic encephalomyelitis in Lewis rats by treatment with myelin basic protein-cell conjugates

Pretreatment of Lewis rats with guinea pig (GP) myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) suppressed the subsequent induction of experimental allegic encephalomyelitis (EAE) with GP-MBP in Freund's complete adjuvant. The degree of suppression correlated positively with the amount of antigen coupled to the SL. GP-MBP coupled to syngeneic red blood cells (RBC) also resulted in suppression of EAE and the extent of the suppression was related to the dose of cells. These regimens of pretreatment also resulted in a decrease in the in vitro lymphocyte proliferative response to GP-MBP and in the extent of perivascular cuffing in the spinal cord. No decrease in the anti-MBP antibody response was detected in rats pretreated with either GP-MBP-SL or GP-MBP-RBC conjugates. Transfer of lymph node cells from rats pretreated with GP-MBP-RBC resulted in a decrease in disease severity in recipients. It is concluded that prior administration of MBP-cell conjugates is an effective way of suppressing the symptoms of EAE.     

A simple and sensitive method for the purification and peptide mapping of proteins solubilized from densonucleosis virus with sodium dodecyl sulfate

An efficient method for the proteolysis and subsequent analysis of dansylated viral (or other) proteins solubilized with sodium dodecyl sulfate (SDS), after their purification using SDS electrophoresis, is described. The dansylation of proteins or the by-products of the reaction do not interfere in this technique. This very simple technique has important advantages over other methods for the purification and characterization of proteins. The method used indicates that the four viral proteins of densonucleosis virus originate at least partially from a common DNA sequence.     

Detection of lipopolysaccharides in polyacrylamide gels by transfer to nitrocellulose followed by immunoautoradiography with antibody and 125I-protein A: "LPS blotting"

Lipopolysaccharides (LPS), which constitute the somatic (O) antigen of gram-negative bacteria, were used to demonstrate the procedure of LPS blotting involving the electrophoretic transfer of electrophoretically resolved LPS from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose filters. Immobilized LPS could then be immunoautoradiographically visualized in situ by reaction with specific anti-LPS antibody and subsequent binding of radioiodinated Staphylococcus protein A. LPS blotting is expected to provide an efficient and specific means of investigating the LPS (O) antigens of gram-negative bacteria.     

The induction of cell-mediated immunity and tolerance to insulin with insulin coupled to syngeneic lymphoid cells

The induction of delayed type hypersensitivity (DTH) and tolerance to DTH against bovine insulin in mice were explored. DTH was induced with insulin in complete Freund's adjuvant (CFA) and was assessed by ear swelling in vivo and by antigen-driven cell proliferation in vitro. Using the concept that thymus cell unresponsiveness is most easily accomplished via antigen on syngeneic membranes, tolerance was induced by iv injection of syngeneic lymphoid cells which had been coupled to insulin with carbodiimide. Mice tolerized with insulin-coupled cells and then sensitized with insulin-CFA had diminished ear swelling in vivo and decreased insulin-driven cell proliferation in vitro. This unresponsiveness was antigen specific but was also inconstant in degree with regard to suppression of ear swelling, most likely because of variability in coupling of insulin to cells. Proliferative responses were more uniformly suppressed, suggesting the possibility that two target cells were being tolerized. Thus, as with other proteins, the biologically active insulin can be used to induce tolerance.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.