Involvement of the neurointermediate lobe of the pituitary gland in the secretion of prolactin and luteinizing hormone in the rat

The relationship between prolactin (PRL) secretion and the neurointermediate lobe (NIL) of the pituitary gland was investigated. Plasma PRL concentrations in rats bearing anterior pituitaries autografted with or without the NIL to the renal capsule were elevated to equal extents at 1 through 6 weeks after surgery (p > 0.10). PRL levels in ovariectomized rats in which the NIL had been removed surgically (NIL-X) or only visualized (NIL-C) were 3–7 ng/ml 4, 7, and 28 days after surgery (p > 0.10); however, they were slightly higher in NIL-X $\text{vs.}$ NIL-C rats 14 days after surgery (p < 0.05). Plasma luteinizing hormone (LH) concentrations in NIL-C rats increased by 36% from 2 to 4 weeks after surgery (p < 0.05); this increase was not detected in NIL-X rats. PRL and LH surges were induced by estradiol implants in ovariectomized NIL-X and NIL-C rats; the profiles of the PRL surges were superimposable, although the magnitude of the LH surge was only 50% that in NIL-C rats (p < 0.05). These results cast doubt on the importance of the NIL in the regulation of PRL secretion either $\text{via}$ secreting hypophysiotropic hormones or $\text{via}$ conducting anterior pituitary hormones directly to the median eminence. However, the NIL may have a physiologically important role in the regulation of LH secretion.     

Suppressor T-cell activity induced as a result of thermal injury.

Many thermally injured patients survive their initial trauma only to succumb to infection at 2 to 4 weeks after the burn. Both clinical and experimental data have suggested that acute thermal insult compromises immune function. In this report we have sequentially examined the ability of thermally injured mice to generate a specific in vitro primary antibody-forming cell (AFC) response to sheep red blood cells (SRBC) at various times after thermal injury. Thermally injured mice appear to lose the ability to generate de novo antibody-forming cells in vitro after thermal injury. The defect was dissected as to the involvement of macrophage (φ), thymus-derived cell (T-cell), or bursal equivalent (B-cell) defects. Murine B cells from burned animals exhibited normal immunological function in the in vitro AFC system. T cells from burned mice were demonstrated as not only dysfunctional in the generation of immune AFC, but also as able to suppress generation of an AFC response by syngeneic normal cells.     

Synthesis of LDH-1 during mamalian oogenesis and early development.

Nuclear multiplication stage embryos were punctured in either the anterior, midlateral, or posterior regions. Both embryos and adults were examined for defects, and the defects were correlated with whether there had been any leakage of cytoplasmic material from the egg at the time of puncturing. Embryonic defects were found, correlated to the site of damage, in all three regions. A number of embryos was followed through development and it was found that 15.1% of the embryos which leaked cytoplasm hatched into larvae, compared to 82.3% of those which did not leak any cytoplasm. Morphological defects arising as a result of lateral puncture only were observed in adults. Many sterile adults were obtained from eggs in which the posterior region had been punctured. The results show that nuclear multiplication embryos are well able to tolerate the disturbance of the cortical cytoplasm created by puncture, but only rarely are they able to compensate for the actual loss of material by regulation. The results were similar to those observed after puncturing Drosophila embryos at the cellular blastoderm stage.     

Aldosterone plasma radioimmunoassay interference by a spirolactone metabolite.

The plasma aldosterone radioimmunoassay developed by Ito et al. was found to be non-specific for aldosterone following administration of the spirolactones, spironolactone and canrenoate-K, in rabbits, dogs and humans. The assay interfering principle was identified as a hydroxylated derivative (M-B) of canrenone, which itself is a metabolite common to both spironolactone and canrenoate-K. The metabolite M-B possessed a high cross-reactivity to the 21-hemisuccinate aldosterone antibody relative to other spirolactones. A modified procedure was developed specific for plasma aldosterone in the presence of M-B. Following single doses of spironolactone and canrenoate-K, aldosterone plasma levels were unchanged in humans and in dogs and decreased in rabbits.     

Intercellular adhesion in the cellular slime mold Polysphondylium pallidum inhibited by interaction of asialofetuin or specific univalent antibody with endogenous cell surface lectin.

Previous work has shown that, as amoebae of the cellular slime mold Polysphondylium pallidum become aggregation competent, they accumulate on their cell surface a carbohydrate-binding protein (lectin) named pallidin. These amoebae also possess cell surface receptors, presumed to contain complex oligosaccharides with a high affinity for the endogenous lectin. If lectin-receptor interactions mediate cell-cell contact, then appropriate concentrations of pallidin inhibitors should block cell cohesion. Two potent macromolecular antagonists of the lectin were employed: the desialylated form of the glycoprotein fetuin and the univalent antibody (Fab) prepared against pallidin. We studied the effects of these inhibitors on rotation-mediated aggregation of P. pallidum amoebae under a variety of assay conditions. Amoebae exposed to hypertonic conditions or to antimetabolites (“Permissive conditions”) were selectively blocked from associating by microgram quantities of the lectin inhibitors, whereas cells in isotonic buffer (“nonpermissive condition”) were only slightly affected. A comparison of the morphology of agglutinates formed under the various conditions allows several explanations for the different susceptibilities to inhibition by antipallidin reagents. Although not conclusive, the work supports a model of cell adhesion in this simple eukaryotic system based at least in part on specific interactions between carbohydrate-binding proteins and receptors on adjoining cells.     

The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.     

Aldosterone plasma radioimmunoassay interference by a spirolactone metabolite

The plasma aldosterone radioimmunoassay developed by Ito et al. was found to be non-specific for aldosterone following administration of the spirolactones, spironolactone and canrenoate-K, in rabbits, dogs and humans. The assay interfering principle was identified as a hydroxylated derivative (M_B) of canrenone, which itself is a metabolite common to both spironolactone and canrenoate-K. The metabolite M_B possessed a high cross-reactivity to the 21-hemisuccinate aldosterone antibody relative to other spirolactones. A modified procedure was developed specific for plasma aldosterone in the presence of MB. Following single doses of spironolactone and canrenoate-K, aldosterone plasma levels were unchanged in humans and in dogs and decreased in rabbits.     

Effects of estradiol benzoate, estrone, and propionates of testosterone or dihydrotestosterone on sexual and related behaviors of ovariectomized rhesus monkeys

Ovariectomized adult rhesus monkeys were injected daily for 10 days with either 1 mg of dihydrotestosterone propionate (DHTP), 1 mg of testosterone propionate (TP), 10 μg of estradiol benzoate (EB), or 500 μg of estrone (El). On the 5th and 10th days of treatment, females received two 24-min behavioral tests with each of two adult males. All females received every hormonal treatment during the course of the study, with the order of treatments counterbalanced. Prior to the initiation of an hormonal treatment, each subject received two tests with no hormone treatment (NORX). Three behaviors related to female proceptivity were recorded. Treatment with DHTP had no influence on any aspect of proceptivity measured, in comparison to the NORX condition, whereas El or TP treatment augmented the frequencies of two of the proceptive behaviors and EB increased all three. The response of the male toward the female was influenced by the female's hormonal condition. Treatment with TP or DHTP did not increase the frequency of male contact or the mount rate in comparison to the NORX condition, whereas EB or El treatment did. In addition, DHTP was the only steroid which failed to increase the percentage of tests with intromission or ejaculation when compared to NORX. Female receptivity, as measured by acceptance or rejection of male contacts, was not different for the NORX-, TP-, EB-, or El-treated conditions. DHTP treatment, however, reduced female receptivity in comparison to all other conditions. Treatment with DHTP or TP resulted in an increase in the frequency of female yawning behavior, whereas neither estrogen treatment showed any effect on this behavior. The influences of TP on female proceptive and male sexual behavior were never duplicated or even approximated by treatment of females with the nonaromatizable DHTP. Nor was there any evidence that TP inhibited female receptivity below the level characteristic of NORX females, as was true for DHTP.     

Hormonal basis of hysterectomy-induced maternal behavior during pregnancy in the rat.

Hysterectomy during the last half of pregnancy (i.e., Day 10–19) induces a rapid onset of maternal behavior; ovariectomy in addition to hysterectomy, prevents this effect. Estradiol and progesterone were tested for their ability to restore short-latency maternal behavior in hysterectomized-ovariectomized (HO) females operated on the 10th, 13th, 16th and 19th days of pregnancy. A single injection of either 20 μg/kg or 100 μg/kg estradiol benzoate (EB) immediately following HO either alone or followed by 0.5 mg progesterone (P) 44 hr later restored short-latency maternal behavior similar to that observed following hysterectomy only. The lower dose of EB was found to be equally effective at all stages of pregnancy and P was unnecessary to induce maternal behavior. The effectiveness of EB in inducing maternal behavior was discussed in relation to the hormonal changes which follow hysterectomy during pregnancy and to those which are associated with the normal onset of maternal behavior around parturition.     

Inhibition of in vitro human chorionic gonadotropin-stimulated testosterone production in testis and of ovulation in the rat by charcoal-treated rat testicular extract

Previously, we described the presence of a factor obtained from a 105,000 X g supernatant of rat testis that was found to inhibit human chorionic gonadotropin (hCG) binding to gonadal receptors. In the present study, similarly prepared testicular extract was tested for its effects on in vitro hCG-stimulated testosterone production by isolated testis interstitial cells and for its effect on spontaneous ovulation in the rat. Incubation of interstitial cells with charcoal-treated extract significantly inhibited the steroidogenic response to hCG in a dose-related manner. This inhibition was also apparent after heating the extract for 10 min at 100 degrees C. Preincubation of the cells with charcoal-treated extract resulted in an inhibitory effect that was not readily reversed by subsequent addition of hCG, revealing an element of irreversibility in the mechanism of inhibition. A single i.p. injection of testicular extract given between 1430-1630 h of proestrus inhibited spontaneous ovulation in the rat. This effect was also observed after heating the extract for 10 min at 100 degrees C; in contrast, no significant effect was obtained with the injection of a similar dose of liver extract. Administration of 5 IU hCG after pretreatment with the testicular extract did not reverse the inhibitory effect on ovulation, indicating that this effect was probably not exerted at the hypothalamus-pituitary level. It is concluded that the aqueous testicular extract contains a factor able to antagonize the physiological events mediated by luteinizing hormone (LH)/hCG, and that this factor is consistent with the presence of an LH/hCG-binding inhibitory activity in rat testis.     

Thermal elution chromatography and the resolution of nucleic acids on hydroxylapatite

Thermal elution chromatography of nucleic acids on hydroxylapatite was studied from a technical standpoint. It is shown that current methods for selecting elution buffers are inadequate. The construction of window diagrams for the purpose of determining suitable conditions is demonstrated. The resolving ability of various buffer-hydroxylapatite systems was studied in some detail. The best system for resolving single- from double-stranded nucleic acids was found to be the use of potassium phosphate together with Bio-Rad HTP (non-DNA grade) which has been preheated in phosphate buffer. Sodium phosphate gives the best resolution among various species of double-stranded nucleic acid.     

Similar properties of [35S]t-butylbicyclophosphorothionate receptor and coupled components of the GABA receptor-ionophore complex in brains of human, cow, rat, chicken and fish

No significant differences are evident in the specific binding characteristics of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to EDTA/water-dialyzed P2 membranes of human, cow, rat, chicken and fish brain. This species similarity includes dissociation constants of 61-77 nM at 37 degrees C, maximum receptor densities of 3-7 pmol/mg protein, and sensitivity to inhibition or displacement by gamma-aminobutyric acid (GABA), two cage convulsants (picrotoxinin and t-butylbicycloorthobenzoate) and the insecticide [1R,cis, alpha S]-cypermethrin, indicating a constancy during vertebrate evolution of the [35S]TBPS binding site and its coupling with other components of the GABA receptor-ionophore complex. As a possible exception, chicken and fish brain membranes appear to be less sensitive than the others to the insecticide alpha-endosulfan. Human and rat preparations are also essentially identical relative to the inhibition of radioligand binding by two GABA mimetics (muscimol and 3-amino-propanesulfonic acid), six other cage convulsants (including examples of three classes of polychlorocycloalkane insecticides), a potent anthelmintic agent (Ivermectin), dimethylbutylbarbiturate, the convulsant benzodiazepine Ro 5-3663, and ethanol. The findings to date with [35S]TBPS and the GABA receptor-ionophore complex in rat brain membranes are therefore generally applicable to human preparations. Cow brain is an appropriate source for large scale preparations in receptor purification studies since it is essentially identical to human and rat preparations in all parameters examined. Species differences in sensitivity to the toxic effects of the convulsants and polychlorocycloalkane insecticides considered are apparently not attributable to receptor site specificity.     

Ergot drugs suppress plasma prolactin and lactation but not aggression in parturient mice

The daily administration of pituitary prolactin (PRL) inhibitors (ergocornine hydrogen maleate and 2-bromo-α-ergocryptine) to parturient Rockland-Swiss Albino mice suppressed circulating levels of PRL and lactation but failed to alter maternal aggression toward adult male intruders. The results suggest that, contrary to popular speculation, PRL may not be necessary for postpartum aggression in the mouse.     

Changes in the binding of copper in the plasma of molybdenum supplemented rats

After incubating plasma of Mo-supplemented rats (Mo-plasma) with ⁶⁴Cu only part of it could be removed by dialysis against EDTA or histidine or by treatment with dithiocarbamate; this nondialyzable Cu was shown to be bound to albumin. The maximal amount of ⁶⁴Cu bound this way equaled the Mo-induced increase in total plasma Cu. After addition of stable Cu dialysis of Mo-plasma against a histidine solution showed that no extra Cu became tightly bound, suggesting that the ⁶⁴Cu binding was due to an exchange between added ⁶⁴Cu and stable Cu already present. Incubating Mo-plasma with Hg compounds prevented ⁶⁴Cu binding and released stable Cu, indicating that Cu in Mo-plasma was sulfhydryl bound. Part of the Mo in Mo-plasma was freely dialyzable. The remaining part was shown to be SH bound as well. The estimated atomic ratio of SH-bound Cu and Mo was unity. Molybdenum increased the number of SH groups in plasma, and for each Cu atom at least one SH group was calculated to be present.     

Chloroplast and cytoplasmic enzymes 1, 2, 3. Subunit structure of pea leaf aldolases.

The pea leaf chloroplastic and cytoplasmic forms of aldolase are very similar in structure. The subunit molecular weights determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate are approximately 37.000. Both aldolases appear to terminate in the same sequence, SerAlaTyrCOOH, and the amino-terminal sequence H₂NGlySerTyrAla was obtained for each. The previously reported differences in kinetic properties and in isoelectric points of the two pea leaf enzymes probably are the consequence of minor differences in amino acid composition or conformation.     

Study of granule reappearance and histamine synthesis in rat mast cells maintained in short-term cultures

A single cutaneous application of components of poison oak or ivy urushiol oils to mice results in contact sensitivity with properties of delayed-type hypersensitivity. The compounds, 3-heptadecylcatechol (HDC, from poison oak urushiol) and 3-pentadecylcatechol (PDC, from poison ivy urushiol) are completely cross-reactive. Covalent bond formation between the o-quinone intermediate of PDC and nucleophilic functionalities such as those found on proteins is known to occur in a regiospecific manner. Amino nucleophiles preferentially attack the 5-position on the catechol ring while thiol nucleophiles attack the 6-position. The present paper describes the immunological properties of the three possible ring monomethylated analogs of PDC. When mice were treated with a single epicutaneous painting with these analogs, only the 5-methyl compound (5-Me-PDC) was found to be an ineffective sensitizer. The 5-Me-PDC analog, however, was capable of inducing cellular proliferation in draining lymph nodes. Furthermore, epicutaneous pretreatment with 5-Me-PDC suppressed the subsequent induction of contact sensitivity to PDC and HDC in an antigen-specific manner. Equivalent treatment with the 6-Me-PDC analog (a good sensitizing agent) resulted in a less consistent and weaker suppressive effect, while the 4-Me-PDC analog did not display any suppressive activity. The suppressive activity could be demonstrated up to 15 days following primary painting with 5-Me-PDC. Lymph node cells obtained from mice 10 days after a single painting with 5-Me-PDC could transfer the suppressive effect. Under certain circumstances 5-Me-PDC could also sensitize, indicating that the analog retains some sensitizing abilities. Hence, blocking the 5-position of the catechol ring results in a major alteration in the type of immune response elicited. Since 5-Me-PDC is specifically blocked in terms of nucleophilic attack by amino groups, it is suggested that attack by amino nucleophiles is of primary importance in the peripheral metabolic processing of these catechols, which in turn determines the outcome of the immune response.     

A direct, stimulating effect of cyclic GMP on purified phosphoribosyl pyrophosphate synthetase and its antagonism by cyclic AMP

The activity of phosphoribosyl pyrophosphate synthetase, purified from a line of rat hepatoma cells in continuous culture, is maximally stimulated (2–4 fold) by less than 10^?7M cyclic GMP. Half maximal stimulation occurs at 2 × 10^?9M. Cyclic GMP stimulates phosphoribosyl pyrophosphate synthetase by decreasing the Km of the enzyme for ATP from 50 μM to 10 μM without affecting the Vmax; it has no effect on the Km for ribose 5-phosphate, the other substrate. Cyclic AMP alone has no effect on the enzyme activity, but at micromolar concentrations it antagonizes the stimulation by cyclic GMP. GMP, GDP, and GTP do not stimulate enzyme activity; and AMP and ADP at micromolar concentrations do not antagonize the effect of cyclic GMP.There is no detectable cyclic nucleotide-activated protein kinase in the enzyme preparation. Cyclic GMP significantly stabilizes the enzyme to heat inactivation. We conclude that cyclic GMP binds directly to the enzyme in an allosteric fashion, causing it to have an increased affinity for one of its substrates, and that cyclic AMP directly antagonizes this effect.     

Reversible inhibition by interferon of the maturation of human peripheral blood monocytes to macrophages

We demonstrated that interferon delays the maturation of human monocytes to macrophages in vitro as assessed by morphologic, histochemical, and biochemical parameters. After exposure to interferon, monocytes were slightly smaller, less stretched out, and had a delayed loss of granules with peroxidase positive reactivity, as compared with control, noninterferon-treated cells. Also, interferon prevented the increase of the specific activity of three lysosomal enzymes (β-galactosidase, β-glucuronidase, and β-N-acetylglucosaminidase) in the monocytes, and enzyme activities were 30–40% of that observed in untreated cells. Both human leukocyte and human fibroblast interferons were effective in delaying the maturation process. The effects of the interferon were species specific and reversible and were neutralized by antiinterferon serum. These studies describe a new nonantiviral effect of interferon, unique in that it involves the delay of maturation of cells in a system which is not associated with cell proliferation. Thus interferon could potentially effect host defense mechanisms and aspects of the immune response which are dependent on the maturation of monocytes to macrophages.     

Changes of plasma dopamine-beta-hydroxylase activity and other plasma constituents during the cold pressor test

The effect of the cold pressor test on plasma DBH activity in ten healthy human subjects was investigated. Parallel changes of other plasma constituents were ascertained as well. Plasma DBH activity rose by over ten per cent in six of the sen subjects and declined by 14 per cent or more in two subjects; the correlations of altertions in DBH activity with changes of high molecular weight plasma constituents were high (r=0.565 to 0.902); correlations with blood urea nitrogen and plasma glucose were low (r=0.002 to 0.248). The results suggest that factors other than neuronal DBH release may be important in alterations of plasma DBH activity following $\text{acute}$ stresses produced by the cold pressor test in man.     

Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: a model for immunodeficiency disease.

The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.