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.     

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.     

Timing and irreversibility of Müllerian duct inhibition in the embryonic reproductive tract of the human male

A study was undertaken to determine (1) the effects of endogenous Müllerian inhibiting substance (MIS) on the developing human fetal genital tract; (2) the time in fetal life when MIS is first capable of inhibiting the growth of the embryonic Müllerian ducts; and (3) the reversibility of the effects of MIS on the developing male Müllerian ducts. Human fetal reproductive tracts were transplanted and grown for sustained periods in vivo in athymic nude mice. The genital tracts from 12 male human fetuses, ages 51 to 68 days postovulation, were grafted without their associated gonads into castrated murine hosts and grown for 30 to 70 days. Controls consisted of genital tracts from 8 female human fetuses, ages day 53 to 70 that were grown under identical conditions. Male specimens grew to approximately one-half the size of female specimens and disclosed varying degrees of inhibition of the Müllerian duct system from absence of the Müllerian ducts in older specimens (after Day 63) to poorly segregated segments of stroma as the mildest defect (less than Day 61). It is concluded that (1) MIS secretion by the embryonic testes probably begins before Day 51 of gestation; (2) the effects of MIS are progressive during the so-called critical window; (3) the effects of MIS are permanent; and (4) the mesenchyme is an important target of MIS.     

Inhibition of murine suppressor cell function by progesterone

The effects of progesterone on murine suppressor cell function generated in allogeneic MLCs were investigated. BALB/c splenic lymphocytes stimulated in vitro with C3H/He cells significantly suppressed the proliferative response of BALB/c lymphocytes in a secondary MLC. This suppression was highly specific for the sensitizing alloantigens since the suppressor cells had no effect on the proliferative response of BALB/c lymphocytes to third-party alloantigens. In addition, BALB/c lymphocytes stimulated with syngeneic cells were observed to nonspecifically suppress the MLC response to a lesser extent. One to 10 micrograms/ml progesterone added at initiation to suppressor cell generating cultures diminished the ability of both alloantigen specific and nonspecific suppressor cell populations to suppress the proliferative response of homologous lymphocytes to alloantigens. Experiments with pyrilamine, an antihistamine, which blocks cytotoxic T lymphocyte (CTL) generation, suggests that progesterone has a direct inhibitory effect on suppressor cell function independent of its ability to block CTL induction. The effects of progesterone on suppressor cells were not due to shifts in peak response time in MLC or induction of radiosensitive cells in progesterone-treated cultures. Estradiol at doses between 5 and 10 micrograms/ml, and cortisol at dose of 1 microgram/ml, also significantly inhibited suppressor cell function. These results suggest that the steroid hormone milieu within the placenta may effect the activity of allogeneic or nonspecific suppressor cell activity.     

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.     

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.     

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.     

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.     

Alpha melanocyte stimulating hormone inhibits prolactin secretion in fetal and infant lambs

The intravenous administration of αMSH (25 μg/kg) to 11 lambs (3 to 29 days of age) suppressed plasma PRL by 15 minutes. The mean basal concentration was 15.3 ± 2.9 ng/ml and the mean nadir was 4.9 ± 0.8 ng/ml (p<0.01). In chronically catheterized fetuses (128–140 days), intravenous administration of αMSH (25 μg/kg) decreased basal PRL levels (89.6 ± 12.4 ng/ml) significantly at 15–30 minutes to levels of 74.3 ± 11.4 ng/ml (p<.01). The degree of suppression of basal PRL levels was less in fetusus (76.9 ± 4.1%) than that induced in the neonates (40.5 ± 7.1%). In younger fetuses <120 days in whom basal PRL levels are low (3.0 ± 2.1 ng/ml), administration of αMSH was without effect. Plasma GH concentrations were not altered by administration of αMSH. The suppression of PRL secretion by αMSH administration could result from increased release of hypothalamic dopamine or be a direct effect on secretion of prolactin by the pituitary.     

Induction kinetics of human suppressor cells in the mixed lymphocyte reaction and influence of prednisolone on their genesis

The induction kinetics of human suppressor cells in mixed lymphocyte cultures (MLC) and the influence of prednisolone on the genesis of these suppressor cells is reported. We induced over 1 to 6 days suppressor cells in one-way MLC (MLC-1), the inhibitory activity of which was tested on a secondary MLC (MLC-2), and on responder cells alone, where lymphocytes were obtained from the same lymphocyte donors as for the MLC-1. In four experiments the degree of inhibition ($\text{x}\text{?}\text{±}\text{SE}$) when suppressor cells were induced for 2, 4, or 6 days was 38.5 ± 11.8, 79.5 ± 7, and 85 ± 6%, respectively, compared to 50.5 ± 9.4, 83.3 ± 7.8, and 85.3 ± 9.8% when 500 ng/ml prednisolone was added to the MLC-1. A similar inhibition pattern was observed when the generated suppressor cells were incubated with responder cells only. The inhibitory activity of these MLC-induced suppressor cells was abrogated by irradiation with 3000 R. Suppressor cells apparently are generated in MLCs between Days 1 and 4; furthermore, their genesis is not affected by usual therapeutic concentrations of prednisolone.     

Inhibition by somatostatin of the proliferation of T-lymphocytes and Molt-4 lymphoblasts

The proliferation of Molt-4 lymphoblasts and phytohemagglutinin-stimulated human T lymphocytes in vitro was inhibited significantly by 10(-12) to 10(-10) M to 10(-13) to 10(-9) M somatostatin, as assessed by the uptake of [3H]thymidine and [3H]leucine, respectively. The inhibitory effect of somatostatin was not attributable to cytotoxicity and was associated with a mean degradation of 52 and over 95% of immunoreactive somatostatin, respectively, after 3 and 24 hr of incubation at 37 degrees C. The specific suppression of Molt-4 lymphoblasts and T lymphocytes by somatostatin represents a distinct mechanism for the specific regulation of immunological responses by neuropeptides of the peripheral nervous system and gastrointestinal tract.     

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.     

Effects of electrical gradients on volume flows across gall bladder epithelium

A volumetric method has been developed which permits continuous registration of volume flows across epithelial tissues. The method was applied to volume flow measurements across rabbit gall bladder epithelium. The rate of fluid reabsorption measured in this way was twice as high as previously observed in sac preparations of the gall bladder. This is probably due to better aeration and stirring of the mucosal solution. It was demonstrated that electrical gradients across the gall bladder induced volume flows towards the negative electrode. In non-transporting bladders volume flows were linearly related with current between 300 and 900 μA in both directions. However, volume flow rates were three times higher from mucosa to serosa than in the opposite direction. From the magnitude of polarization potentials, observed after switching off the current, the conclusion was reached that all of the current-induced volume flow is an osmotic flow due to salt polarization in the unstirred layers of the tissue. By implication, so-called streaming potentials observed during osmotic flows reflect solely polarization effects. In actively transporting gall bladders a 200 μA current increased or decreased the flow rate twice as much as expected from polarization effects alone. Therefore passage of current interfered directly with the active transport mechanism of gall bladder epithelium.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A₁, A₂, E₁, E₂, F_2α and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂. All the prostaglandins (except PGF_2α and its 15-keto-metabolites) relaxed the tissue. However, only PGE₁, E₂, and their 13,14-dihydro-metabolites, were effective at concentrations below 10⁻⁸ M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

Different sensitivities of acetylcholine-induced “after-hyperpolarization” compared to dopamine-induced hyperpolarization,to ouabain or to lithium-replacement of sodium,in rabbit sympathetic ganglia

The ganglionic potentials elicited by a test dose of acetylcholine (ACh) acting nicotinically consist of an initial depolarization followed by a longer “after-hyperpolarization” (after-HP). The after-HP is abolished selectively and relatively rapidly after treatment with ouabain or with Li-replacement of 90% of Na in Ringer. The hyperpolarizing response to a test dose of dopamine (DA), however, is as or more resistant than the ACh-depolarization to either treatment. These findings provide further strong evidence that the slow inhibitory postsynaptic potential (s-IPSP), for which DA is the transmitter in these ganglia, is $\text{not}$ mediated by an activation of an electrogenic NaK pump.     

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.     

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.     

Collagen in the egg shell membranes of the hen

Collagen-like proteins have been found in the egg shell membranes of the hen. Materials similar to types I and V collagens were detected in each of the two layers of this membrane, the thick outer membrane and the thin inner membrane. Collagen was extracted by acid-pepsin digestion and isolated by differential salt precipitation. Identification of type-specific collagen-like material was established by coelectrophoresis on SDS-polyacrylamide gels using known collagen standards. These bands were susceptible to digestion by bacterial collagenase. From differential staining of the gels it was estimated that the ratio of collagen types I:V was approximately 100:1. Further confirmation of these biochemical results was obtained with immunofluorescence microscopy using type-specific antisera against chicken types I and V collagen with the indirect sandwich technique. Both the inner and outer shell membranes contained the two types of collagen. Within each membrane, the large, coarse 2.5-micron fibers contained predominantly type I collagen-like material, while type V collagen was mainly associated with the delicate narrower fibers of approximately 0.6-micron diameter. These tended to be concentrated in the inner membrane. At the electron microscopic level, both types of fibers were coated with glycoproteins that stained positively with ruthenium red. The deposition of these collagen-like substances by the hen oviduct on to the surface of the developing egg is an additional example of interstitial-type collagen synthesis and secretion by epithelial rather than by mesenchymal cells.     

Regulation of aldosterone synthesis

The effects of angiotensin II and ACTH on cyclic AMP and aldosterone synthesis were studied in cells isolated from the bovine adrenal cortex. Angiotensin is a more potent stimulus of aldosterone synthesis than ACTH and the action of ACTH on aldosterone synthesis in cells from the glomerulosa is augmented by the presence of cells from the fasciculata. Angiotensin stimulates aldosterone synthesis in the absence of detectable changes in cyclic AMP, but the cells do respond to dibutyryl cyclic AMP leaving open the possibility that a cyclic nucleotide may play a role in the steroidogenic action of this hormone in the outer zone of the bovine adrenal cortex.