Cultivation and characterization of macrophages from murine embryonic skin. Are they Langerhans cells?

We have observed a population of trypsin-resistant adherent cells in long-term primary cultures of murine embryonic skin. These cells were subsequently demonstrated to share a variety of characteristics with cells of the monocyte/macrophage lineage. The trypsin-resistant adherent cells stained positively for nonspecific esterase, exhibited surface receptors for Fc-IgG, and complement components as well as strong phagocytic activity. Additionally, these cells exhibited membrane ATPase enzyme activity and a large proportion of the cells expressed la antigens as detected by cytotoxicity and membrane fluorescence. The possible relationship between these trypsin-resistant adherent cells and Langerhans cells of the skin is discussed.     

Glutathione biosynthesis in murine L5178Y lymphoma cells

The pyruvate dehydrogenase complex from pea leaf mitochondria was rapidly deactivated in the presence of 50 to 200 μm ATP. The deactivation of the complex requires Mg²⁺ as shown by EDTA inhibition of deactivation. Deactivation was inhibited by 0.1 to 1 mm pyruvate or dichloroacetate. Activation required 10 mM Mg²⁺ or Mn²⁺ but Ca²⁺ and K⁺ had no effect. Activation was inhibited by the phosphatase inhibitor, F^?. Autoradiograms of nondissociating electrophoresis gel, crossed immunoelectrophoresis gels, and dissociating sodium dodecyl sulfate electrophoresis gels of the complex showed that one protein is labeled. Labeling of this protein is prevented by Mg²⁺, pyruvate, and dichloroacetate. The pyruvate dehydrogenase complex was isolated in a partially deactivated state and reactivation required exogenous Mg²⁺ and was inhibited by F^?. These results are taken as conclusive evidence that the pyruvate dehydrogenase complex in pea leaf mitochondria undergoes interconversion between deactivated and activated states by covalent modification (phosphorylation-dephosphorylation) catalyzed by a kinase and phosphatase. Isolation of the complex in a partially deactivated (phosphorylated) state suggests a physiologically significant role for this regulatory mechanism.     

Generation of nonspecific murine cytotoxic T cells in vitro by purified human interleukin 2

Murine spleen cells developed into nonspecific cytotoxic cells within 72 hr of culture in the presence of highly purified sources of human interleukin 2. In whole spleen cell cultures, human interleukin 2 generated effector cells which were Thy 1.2⁺, Lyt 2.2⁺, resistant to γ irradiation (1000 R), and capable of lysing both H-2 compatible and incompatible targets. The effector cells generated in this manner were not restricted to classical natural killer cell-sensitive targets. If thymus-derived cells (T cells) were depleted from the spleen cell population before culture with human interleukin 2, the effector cells generated were enriched in effectors capable of lysing natural killer cell-sensitive targets. Interferon was not produced in interleukin 2-stimulated spleen cell cultures. In addition, heterologous antibody to murine -γ-interferon did not abrogate the generation of cytotoxic cells by human interleukin 2. These and additional data suggest that human interleukin 2 is capable of stimulating γ-irradiation-sensitive Thy 1.2⁺ cell(s) capable of lysing a variety of target cells regardless of inherent sensitivities to classical natural killer cells. Thy 1.2^? cells were also stimulated by human interleukin 2 and lysed only natural killer cell-sensitive targets. Human interleukin 2 caused some Thy 1.2^? cells to become susceptible to lysis by anti-Thy 1.2 serum and complement.     

Onset of the receptive and proceptive components of feminine sexual behavior in rats following the intravenous administration of progesterone

The present study was carried out in order to assess the time course of action of progesterone (P) in the facilitation of complete feminine sexual behavior. Female rats (estrogen primed via 5% E₂ Silastic capsules) were given 200 μg of P either intravenously (iv) or subcutaneously (sc), and tested for estrous behavior at $\text{1}\text{4}$, $\text{1}\text{2}$, 1, 2, and 4 hr after treatment. Among iv-treated animals, significant amounts of lordosis behavior were seen as early as $\text{1}\text{2}$ hr, and a dramatic rise in solicitation behavior was observed at 2 hr. Although sc-treated animals displayed significant amounts of lordosis and solicitation behavior at 2 hr, the behavior was not maximal until 4 hr. Intravenous administration of 400 μg P was equipotent to 200 μg P, whereas 50 μg of iv P was relatively ineffective. A dual mechanism hypothesis pertaining to progesterone's actions in the facilitation of both the receptive and preceptive components of feminine sexual behavior in rats is discussed.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.     

Unsaturated fatty acids as second messengers of superoxide generation by macrophages

Chemically elicited guinea pig peritoneal exudate macrophages respond by superoxide (O2-) production to a large number of unrelated stimulants. It has been found that 8 out of 10 stimulants also induce arachidonic acid (20:4) liberation and thromboxane synthesis. The elicitation of O2- production by most stimulants was reduced or totally suppressed by three procedures that inhibit the activity of endogenous phospholipases: the use of drug p-bromophenacyl bromide, elevation of the cellular cyclic AMP level, and the removal of extracellular Ca2+. O2- production in response to concanavalin A, wheat germ agglutinin, and fMet-Leu-Phe were exquisitely sensitive to inhibition of phospholipase activity. Exogenously applied 20:4 as well as other unsaturated fatty acids (linolenic, linoleic, and oleic) induced massive and instantaneous O2- production in a dose-dependent manner. Saturated fatty acids (stearic) and methyl esters of unsaturated acids were inactive. Lysophosphoglycerides were also inactive. Incubation of macrophages with inhibitors of cyclooxygenase or lipoxygenase did not prevent the elicitation of O2- production by stimulants or fatty acids. On the contrary, O2- formation was enhanced by indomethacin and indomethacin by itself was capable of evoking O2- generation. Treatment of 20:4 with soybean lipoxygenase did not abolish its capacity to induce O2- production; native and lipoxygenase-treated 20:4 exhibited similar dose-response ratios. Purified 15-hydroxyeicosatetraenoic acid also elicited O2- production by macrophages with a potency comparable to but not exceeding that of 20:4. Equimolar amounts of prostaglandin E2 were inactive. These findings suggest that liberation of unsaturated fatty acid (principally, 20:4) from membrane phospholipids, as a consequence of phospholipase activation, is a necessary step in the elicitation of an oxidative burst in macrophages. O2- generation is stimulated by unesterified 20:4 and, possibly, by certain metabolites of 20:4. It appears that the lipoxygenase pathway may generate metabolites with stimulating capacity while the cyclooxygenase pathway is abortive.     

Preparation of lymphocyte-activating factor from continuous murine macrophage cell lines

Lymphocyte-activating factor (LAF), a mitogen for thymocytes and T lymphocytes, is released into the culture medium by human mononuclear cells and mouse peritoneal exudate cells following treatment with various macrophage stimulants. Experiments were performed to determine if recently described mouse macrophage cell lines released LAF in response to stimulation with bacterial lipopolysaccharide. Four continuous cell lines (P388-D₁, J774, WEHI 3, and PU5-1.8) were found to release LAF in serum-free medium following endotoxin stimulation. The results of partial purification indicated that LAF obtained from cell lines had a higher molecular weight and lower isoelectric point than LAF from human mononuclear cells.     

Glucocorticoid binding to solubilized components of human placental trophoblast membranes

We have recently described glucocorticoid uptake by human placental membrane vesicles which is specific, saturable and has a low K (7 nM). This paper describes solubilization of these vesicles with Triton x-100 and successful demonstration of glucocorticoid binding to the putative transport site. This was accomplished by analysis of corticosterone binding to the 140,000 × g supernatant of solubilized vesicles using G-75 Sephadex chromatography. The amount of bound corticosterone present in the void volume was proportional to the concentration of solubilized vesicles. The specificity of binding was tested by coincubation of tritiated corticosterone with 100-fold excesses of various steroids. The relative ability of various steroids to inhibit binding was corticosterone=pregesterone- >aldosterone. Triamcinolone acetonide, and estradiol were ineffective competitors. We conclude from these studies that human placental membranes contain glucocorticoid-specific binding sites which can be solubilized with Triton x-100. It is possible that these sites are involved in membrane mediated transport of glucocorticoids by this tissue.     

Activation of a Na+-dependent amino acid transport system in preimplantation mouse embryos

The uptake of l-methionine-methyl-³H and l-leucine-³H from completely defined medium into acid-soluble fractions of preimplantation mouse embryos has been studied. Late four-cell embryos and early blastocysts raised in vitro can concentrate both amino acids by processes which exhibit saturable, Michaelis-Menten type kinetics, characteristic of carrier-mediated active transport systems. This uptake is temperature-sensitive and inhibited by certain amino acids which compete for the same uptake sites. Methionine uptake seems to be mediated by a single transport system (K_m = 6.25 × 10^?5M) at the four-cell stage. Complex kinetics suggest that two distinct transport systems exist at the early blastocyst stage (K_m = 6.25 × 10^?5M; 8.9 × 10^?4M). V_max values (mg/embryo/15 min) for methionine and leucine transport increase significantly from the late four-cell stage to the blastocyst stage, suggesting that additional carriers are produced or activated during development.Most importantly, leucine and methionine transport is Na⁺-independent at the four-cell stage, methionine transport is partially dependent at the morula stage, and both amino acids are completely Na⁺-dependent at the blastocyst stage. The cumulative results suggest that preimplantation embryos accumulate leucine and methionine by specific, chemically mediated, active transport systems. The qualitative and quantitative developmental changes in cell membrane function may represent preparatory steps for subsequent growth of embryonic and/or trophoblastic cells.     

Questioning of reported evidence for guanosine tetraphosphate synthesis in a ribosome system from mouse embryos.

In 1974, Irr, Kaulenas and Unsworth reported that ppGpp is synthesized by cytosolic ribosomes from mouse embryos and proposed a role for ppGpp in the process of differentiation. This proposal is being challenged because ribosomes of mouse embryos from various stages of development and of mouse embryoid bodies were completely inactive in ppGpp formation.     

Biosynthesis of phytoene from isopentenyl pyrophosphate by a Neurospora enzyme system.

An enzyme system catalyzing the conversion of isopentenyl pyrophosphate to phytoene has been isolated from Neurospora crassa mycelia. This enzyme system shows an absolute requirement for Mg^?, but no other cofactors. Cultures of N. crassa exhibit a low level of phytoene synthesizing activity when grown in the dark. A 2-min in vivo blue light irradiation results in a ninefold increase in activity after 24 h. This increase is dependent on the duration of the light treatment and is inhibited by cycloheximide. A similar blue light-induced elevation of phytoene synthesizing activity was demonstrated in an albino-1 mutant. This enzyme activity was not found in either dark-grown or irradiated cultures of an albino-2 or an albino-3 mutant.     

Role of hypothalamic serotonergic receptors in the control of lordosis behavior in the female rat

The role of serotonin in mediating hypothalamic control of sexual behavior in estrone-primed ovariectomized (OVX) rats was studied by comparing the lordotic patterns following medial preoptic (MPOA) and arcuate-ventromedial (ARC-VM) infusions of serotonin (5-HT), methysergide (MS), and vehicle. In the initial experiments, low receptivity (preinfusion receptivity: mean lordosis/mount ratio = 0.164) was maintained by priming each animal with a low dose of estrone 48 hr prior to mating. The infusion of MS in either the MPOA or ARC-VM area resulted in a significant enhancement of lordotic behavior from initial low receptivity, 5-HT infusions were found to have no statistically significant effect upon lordotic behavior. In order to corroborate the findings observed in the low preinfusion receptivity protocol, OVX rats were primed with higher doses of estrone to maintain a high level of receptivity (preinfusion receptivity: mean lordosis/mount ratio = 0.787). Using this protocol, significant depressions in lordotic behavior were observed following MPOA or ARC-VM infusions of 5-HT, It was thus proposed that serotonergic receptors within the MPOA or ARC-VM areas have inhibitory effects upon lordotic behavior. In addition to the effects of 5-HT upon estrogen-induced sexual receptivity, serotonergic influences upon luteinizing hormone-releasing hormone (LRH)-facilitated mating behavior were also evaluated. Comparisons were made between the lordotic responses following MPOA or ARC-VM infusions of vehicle, LRH, or LRH with 5-HT in OVX rats primed with low doses of estrone. The infusion of LRH into the MPOA or ARC-VM significantly enhanced lordotic behavior above vehicle levels. However, the addition of 5-HT to the LRH infusate abolished this behavioral enhancement. These findings indicated that LRH and 5-HT have opposing effects within forebrain areas known to be important for the control of lordotic behavior.     

On the role of the collagen carbohydrate residues in the platelet: Collagen interaction

It has been proposed that the platelet : collagen interaction is mediated in part by the collagen carbohydrate residues. To rest this hypothesis we have oxidized monomeric and polymeric collagen with sodium periodate under conditions specifically designed to minimize destruction of periodate-susceptible bonds other than in the carbohydrate residues. Oxidation of the collagen significantlly reduced its ability to interact with platelets. The extent of inhibition paralled the extent of carbohydrate destruction. Oxidation with periodate also delayed the polymerization of the monomeric collagen, but even after polymerization the oxidized collagen failed to initiate the release reaction. These observations suggest that the collagen carbohydrate residues may be either near to or part of the site(s) on the collagen molecule required for platelet adhesion.     

The ventromedial nucleus of the hypothalamus and the hormonal arousal of sexual behaviors in the female rat

Bilateral lesions of the ventromedial nucleus of the hypothalamus interfered with the estrogenic induction of sexual receptivity in the female rat, but seemingly did not affect the ability of female rats to show lordosis following combined stimulation with estrogen and progesterone. In addition, ventromedial hypothalamic lesions did not affect the ability of females to show male-like sexual activity in response to exogenous androgenic stimulation.     

ESR analysis of the FAD in bovine liver monoamine oxidase

Two hydrazine spin labels, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carbonyl ethyl hydrazine and 1-oxyl-2,2,6,6-tetramethylpiperidino-4-hydrazine, were synthesized as probes of the FAD binding site of monoamine oxidase. The reporter nitroxide moiety showed an ESR spectrum classified as partially immobilized which is indicative of FAD near the surface of the enzyme. Attempts to pick up flavin semiquinone or free radical intermediates during substrate oxidation with the spin traps 5,5-dimethyl-1-pyrroline-1-oxidase and phenyl-t-butylnitrone were not successful.     

On the mechanism of production of tandem genetic duplications in phage lambda

We have asked whether the mechanism by which tandem genetic duplications arise in the chromosome of phage lambda is inter- or intramolecular. Two parental phages carrying genetic markers at opposite ends of the phage chromosome have been grown in mixed infection, and progeny phages carrying newly-arising tandem duplications have been analysed to determine whether they carry the markers in parental or recombinant configuration. Ordinary genetic recombination of the markers has been prevented by mutations in the phage and host. Phages carrying tandem duplications are isolated by use of CsCl density gradients and an Escherichia coli strain that does not plate deletion phages. Of the duplication mutants isolated under these conditions, 13% carry the input markers in recombinant configuration. This suggests that tandem duplications can be produced via an intermolecular route which joins sequences originally present on different DNA molecules.     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.     

Inorganic phosphate estimation in the presence of dithiothreitol

Dithiothreitol (Cleland's reagent) is widely used as a sulfhydryl protective reagent in biochemical systems in vitro. For example, dithiothreitol has been used to achieve maximal rates of enzyme activity for protein phosphokinase reactions (1–4) as well as for phosphoprotein phosphatese assays (1,5). Meisler and Langan (5) have utilized ³²P-labeled histone phosphoprotein as a substrate to examine the protein phosphatase activity of a rat liver cytosol enzyme preparation. However, if one is dealing with a phosphoprotein substrate which may not be labeled with ³²P, it would be desirable to measure the phosphatase activity using a sensitive chemical analysis, e.g., the method of Berenblum and Chain (6) as modified by Martin and Doty (7). We have been interested in examining the protein phosphatase activity associated with prostatic chromatin and the androgenic influences thereupon, using nonhistone and histone phosphoproteins and phosvitin as substrates (Ahmed and Davis, unpublished data). In designing these experiments, 1–3 mm dithiothreitol was added in the reaction medium; this subsequently resulted in interference of P_i analysis using the Berenblum and Chain procedure (6,7). We have, therefore, systematically examined the conditions which may be used to assay P_i when dithiothreitol is present in the sample. The following report deseribes these observations.     

Translation of avian myeloblastosis virus RNA in a reticulocyte cell-free system.

AMV-RNA was translated into a precursor polypeptide of 76,000–80,000 daltons in a reticulocyte cell-free system. Besides this high molecular weight precursor, several smaller precursor polypeptides and the four major internal structural viral proteins were also synthesized. These virus-specific translation products were detectable after immunoprecipitation with antiserum against the gs-antigens of AMV.