The effects of extrahypothalamic cycloheximide on sexual receptivity in the rat

The present experiment was concerned with extrahypothalamic control of sexual receptivity. Cycloheximide, an inhibitor of protein synthesis, suppressed sexual receptivity in the steroid-primed ovariectomized rat when it was injected into the preoptic area. Cyclohexamide was without effect when injected into the cortical and medial nuclei of the amygdala, lateral septum or caudate nucleus.     

Diurnal variations in activity of four pyridoxal enzymes in rat liver during metabolic transition from high carbohydrate to high protein diet.

The diurnal variations in enzyme activities including tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT) and serine dehydratase (SDH) have been studied in rats trained to a 2 hour meal feeding schedule (″2+22″) during metabolic transition from 12.5 to 60% protein diets over a period of 21 days. Although the maximal TAT activity on the first day was slightly lower compared with other days, both TAT and ODC activities adapted rapidly to the increased dietary protein from the first day. The responses of TAT and ODC to the food were so rapid that the maximal value was observed only 4 hrs after the onset of feeding. After each feeding ODC activity decreased rapidly after 4 hours, while TAT activity declined only after 6 hours had elapsed. No clear diurnal rhythm was observed in either OAT or SDH, though OAT activity tended to decrease from the beginning of the dark period and to resume a slow adaptation after about four hours. In contrast to ODC and TAT both OAT and SDH required about 7 days to fully adapt to the high protein diet. The activities of the four enzymes were also compared after 4 groups of rats had been adapted to the ″2+22″ feeding of 12.5, 30 and 60% protein diets and to 60% diet, $\text{ad}$$\text{libitum}$, respectively. The enzyme activities were not directly proportional to the protein content of the diets although higher activity was observed on the high protein diets. The diurnal variations in both TAT and ODC were observed in all ″2+22″ groups although the timing of the peak values were slightly different from each other. The maximal activities of TAT were found at earlier times in 12.5 and 30% protein groups than in the 60% protein group. The peak time for ODC activity was found at a later time in the 12.5% protein group than in rats fed 30% and 60% protein. $\text{Ad}$$\text{libitum}$ rats fed 60% protein maintained relatively high levels of TAT activity compared to the rats on the schedule. However, the maximal activity of ODC on the 60% ″2+22″ protein diet $\text{ad}$$\text{libitum}$ was so low that a diurnal rhythm was not clearly evident.     

Cholinephosphophotransferase activities in early rat embryos and their associated placentas.

CDP-Choline:1,2-diglycerolcholinephosphotransferase (EC 2.7.8.2, cholinephosphotransferase) activities were determined in subcellular fractions prepared from rat embryos, placentas, or yolk sacs obtained on the fourteenth day of gestation. It was found that, in all of the tissues studied, cholinephosphotransferase activity (1) copurified with NADPH-cytochrome c reductase activity (EC 1.6.2.4), (2) was maximal around pH 8.0; (3) was stimulated by MgCl₂, exogenous diolein, and cytidine diphosphocholine (CDP-choline); and (4) was highest in homogenates of placentas, lowest in those of embryos, and intermediate in those of yolk sacs. These data substantiate, for the first time, that the early mammalian (rat) embryo, placenta, and yolk sac have the ability to synthesize phospholipids de novo.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

Optical measurements of intracellular oxygen concentration of rat heart in vitro

The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a₃ at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a₃ at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O₂ (P₅₀) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O₂ titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a₃ as intracellular O₂ indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a₃, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O₂ affinities between myoglobin and cytochrome a + a₃. The results indicate that there may be an O₂ gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a₃ can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a₃ is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.     

A sterile leakproof plastic vial for cell cryopreservation in liquid nitrogen: Application to parasitic protozoa

Polyethylene bulbs derived from sterile transfer pipets were evaluated as leak-proof vials for cryopreservation of parasitic protozoa. The bulbs were easily frozen and stored in conventional liquid nitrogen refrigeration equipment and were less expensive and safer to handle than plastic screw-capped cryovials. The easily testable heat seal of the polyethylene bulb never failed during longterm immersion in liquid nitrogen, and dispensing of thawed material was simple and safe. Good survival of L. donovani (80%), T. rhodesiense (95%), and C. fasciculata (95%) was obtained after cryopreservation in polyethylene bulbs or plastic screw-capped vials. The polyethylene bulb was considered an excellent container for the safe cryopreservation of pathogens and other biological material.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. III. Cholera enterotoxin induces lymph node cells to release soluble factor(s) which enhance(s) antibody synthesis by antigen-treated lymph node cells.

Unprimed or KLH-primed rabbit lymph node cells were pulsed with cholera enterotoxin or KLH for 2 hr and washed. KLH-treated LNC were mixed with equal numbers of CT-treated LNC or boiled CT-treated LNC. Cocultivation of CT-treated LNC with KLH-treated cells resulted in at least a 100% increase in antibody synthesis compared to control cultures. Delaying cocultivation for 24 hr reduced enhancement to 25%. Thus it appears that an early event—before 24 hr—is involved in CT enhancement. Using ¹²⁵I-CT, it was shown that these effects were not due to CT carry-over. When KLH- and CT-pulsed LNC were cultured in chambers separated by polycarbonate membranes (0.2- to 0.4-μm pore size) antibody production was enhanced 50–80%. Supernates of CT-treated LNC also enhanced antibody production by KLH-treated LNC. These results suggest that CT triggers the release of soluble factor(s) which enhance(s) antibody synthesis by antigen-primed and antigen-challenged LNC.     

Induction of male-typical aggression by androgens but not by estrogens in adult female mice

Ovariectomized adult CF-1 female mice were implanted with silastic capsules containing either testosterone (T), dihydrotestosterone (DHT), methyltrienolone (R1881), estradiol (E2), diethylstilbestrol (DES), or oil vehicle and were tested for aggressive behavior. The androgenic treatments (T, DHT, R1881) were highly effective in promoting male-like aggression while the estrogens (DES, E2) were completely ineffective. Subsequent receptor-binding studies confirmed assumptions about the specificity of DES, DHT, and R1881 binding to estrogen and androgen receptors in mouse hypothalamus.     

Studies on the relationship between glycolysis, lipogenesis, gluconeogenesis, and pyruvate kinase activity of rat and chicken hepatocytes.

Chicken hepatocytes synthesize glucose and fatty acids at rates which are faster than rat hepatocytes. The former also consume exogenous lactate and pyruvate at a much faster rate and, in contrast to rat hepatocytes, do not accumulate large quantities of lactate and pyruvate by aerobic glycolysis. α-Cyano-4-hydroxycinnamate, an inhibitor of pyruvate transport, causes lactate and pyruvate accumulation by chicken hepatocytes. Glucagon and N⁶,O²′-dibutyryl adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) convert pyruvate kinase (EC 2.7.1.40) of rat hepatocytes to a less active form. This effect explains, in part, inhibition of glycolysis, inhibition of lipogenesis, stimulation of gluconeogenesis, and inhibition of the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment by these compounds. In contrast, pyruvate kinase of chicken hepatocytes is refractory to inhibition by glucagon or dibutyryl cyclic AMP. Rat liver is known to have predominantly the type L isozyme of pyruvate kinase and chicken liver predominantly the type K. Thus, only the type L isozyme appears subject to interconversion between active and inactive forms by a cyclic AMP-dependent, phosphorylation-dephos-phorylation mechanism. This explains why the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment of chicken hepatocytes is insensitive to cyclic AMP. However, glucagon and dibutyryl cyclic AMP inhibit net glucose utilization, inhibit fatty acid synthesis, inhibit lactate and pyruvate accumulation in the presence of α-cyano-4-hydroxycinnamate, and stimulate gluconeogenesis from lactate and dihydroxyacetone by chicken hepatocytes. Thus, a site of action of cyclic AMP distinct from pyruvate kinase must exist in the glycolytic-gluconeogenic pathway of chicken liver.     

Cyclic AMP phosphodiesterase activity at the external surface of intact skeletal muscles and stimulation of the enzyme by insulin

Small intact frog skeletal muscles were exposed to radioactively labeled adenosine 3′,5′-cyclic monophosphate (cAMP) during incubation in frog Ringer's solution buffered with Tris (RT). The fate of the nucleotide was followed by measuring the products in the incubation media. Paper chromatography was used for the separation and identification of these products; the amounts were measured using liquid scintillation spectrometry. It was found that cAMP was degraded to AMP, which was then converted to IMP and, to some extent, inosine. The degradation of cAMP to AMP was markedly inhibited by theophylline (10 mM) suggesting the presence of cAMP phosphodiesterase activity at the muscle surface. Kinetic studies of enzyme activity in situ revealed two apparent K_m values: 0.33 μm and 55 μm. Insulin (0.3 unit/ml) increased the phosphodiesterase activity at concentrations of cAMP ranging from 2 to 17 μm. The possible roles of the surface phosphodiesterase were discussed.     

Use of a DNA probe for mapping by electron microscopy the ribosomal sequences in ribosomal RNA precursors from duck cells

This report describes the use of purified ribosomal DNA to map by electron microscopy the relative positions of the 18 S and 28 S RNA regions within the duck rRNA precursor and their relationship to the non-conserved portions of the precursor molecule. By repeated fractionation of the total DNA, based on the relative reassociation rates of the DNA sequences with different degrees of repetition, a fraction of the rapidly renaturing DNA was obtained which comprised only 6% of the total DNA, but contained 71% of the rRNA cistrons. Further purification of the rDNA was achieved by saturation hybridization with rRNA and separation of the rRNA-rDNA hybrids by banding in CsCl. In this manner, an rDNA-rRNA fraction was obtained which had a buoyant density of 1.805 g/cm³, an RNA to DNA ratio of 1.01, and a base composition for the RNA present in the hybrid identical to that of an equimolar mixture of 18 S and 28 S rRNA. The final yield of rDNA isolated by this procedure is 32%. When the purified rDNA was annealed with a mixture of 18 S and 28 S rRNA and the hybrids spread for electron microscopy, they appeared as two distinct populations with a number-average length of 0.62 ± 0.13 μm and 1.37 ± 0.18 μm, respectively. Likewise, hybrids between the rRNA precursor, isolated from duck embryo fibroblasts, and the rDNA appeared as structures containing two duplex regions of lengths 0.60 ± 0.11 μm and 1.38 ± 0.15 μm, separated from each other by a single-stranded region appearing as a large bush: this represents a portion of the precursor molecule not conserved during processing of the parent molecule. From these observations a model of the structure of the duck rRNA precursor is proposed.     

Cholesterol autoxidation: identification of the volatile fragments.

The volatile fragments of air-aged cholesterol were analysed by means of gas chromatography-mass spectrometry; The following fourteen compounds were identified: ethanol, acetic acid, acetone, 2-methylpropene, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-butanone, 2-methylpropionic acid, 2-methyl-2-butanol, 2-pentanone, 3-methyl-2-butanone, 2-methyl-1-pentene, 2-methyl-2-pentanol, and 2-methyl-4-penten-2-ol. Their formation via decomposition of initially formed sterol hydroperoxides is discussed.     

Separation of enzymatic activities in Dictyostelium discoideum by high-pressure gel permeation chromatography.

High-pressure gel permeation chromatography was used to separate the cyclic AMP phosphodiesterase and ATP pyrophosphohydrolase activities of Dictyostelium discoideum. Two types of column packings, with different functional groups on the silica-bonded carbon side chains, were used to separate the two activities in approximately the same amount of time and with the same elution pattern. Recovery of both activities was enhanced when acetate, rather than sulfate, was the mobile phase. This recovery of activity following chromatography at high pressure demonstrates that high-pressure gel permeation chromatography can be used for the purification of enzymes.     

Human eosinophil stimulation promoter lymphokine: Production by antigen stimulated lymphocytes and assay with a new electro-optical technique

A human lymphokine, eosinophil stimulation promoter (ESP), was shown to be produced by mononuclear leukocytes in response to non-helminthic antigens. Supernatants of tuberculin-stimulated cultures of mononuclear leukocytes from tuberculinskin test reactive humans contained non-dialyzable ESP activity; ESP could not be demonstrated in the supernatants of tuberculin stimulated cultures from skin test negative donors. Thus, human ESP, like murine ESP, is a soluble lymphokine product of sensitized mononuclear cells. ESP activity was also demonstrated in supernatants of mononuclear leukocyte cultures stimulated with streptococcal antigens. ESP selectively enhanced migration of eosinophils. ESP production could be blocked by puromycin, and its activity was diminished by prior incubation with eosinophil-rich granulocytes. Correlation of antigen stimulated ESP activity with skin test reactivity establishes ESP as another in vitro correlate of delayed hypersensitivity in man. A video method of data collection and analysis is described which greatly facilitates in vitro studies of ESP activity. The method is applicable to the study of other lymphokines which effect cellular migration.     

Inhibition of hepatic gluconeogenesis by dichloroacetate

Gluconeogenesis from lactate by isolated hepatocytes suspended in a low bicarbonate medium is effectively inhibited by the hypoglycemic agent dichloroacetate. With this medium dichloroacetate suppresses the accumulation of the components of the malateaspartate shuttle, limits mitochondrial utilization of cytoplasmic reducing equivalents, and makes the availability of pyruvate and/or oxaloacetate limiting for gluconeogenesis. Much less inhibition is observed with hepatocytes suspended in a medium (Krebs?Henseleit saline) containing physiological concentrations of bicarbonate. No inhibition is observed with Krebs-Henseleit saline supplemented with lysine as a source of amino groups for the malate-aspartate shuttle. Thus, dichloroacetate inhibition of gluconeogenesis is observed only when hepatocytes are incubated in a medium deficient in bicarbonate and amino acids. This means that the action of dichloroacetate as a hypoglycemi agent is best explained by stimulation of peripheral tissue utilization of glucose and potential precursors for hepati gluconeogenesis rather than by direct inhibition of hepatic gluconeogenesis.     

Inactivation of staphylococcal enterotoxin A by chemical modification

(3-¹⁴C)-serine was fed through cut ends of 10-day-old shoots of normal (Ganga-2) and opaque (opaque-2) maize ($\text{Zea}\text{mays}$ L.) and allowed to be metabolized in light for 24 hours. Subsequent methanolic extraction, radiochromatographic separation and radioautographic analysis showed that 10.4 and 10.3 percent of the absorbed ¹⁴C-serine activity was incorporated into L-tryptophan by the shoot tissues of the normal and opaque maize respectively.By coupling extraction of the enzyme with ammonium sulfate precipitation (75% saturation) it was possible to show a serine-dependent indole utilization by the enzyme preparations from coleoptiles (4-day-old), kernels (18 and 28 days after anthesis) and first nodal callus tissues (one-month-old) of the two corn varieties. Furthermore, the indole disappearance from reaction mixture occurred with a concomitant transfer of (3-¹⁴C)-serine appearance into tryptophan.     

Increase in fluorescence energy transfer across lipid bilayers induced by valinomycin

The translocator antibiotic, valinomycin, increases the energy transfer between fluorophores across a lipid bilayer membrane, contrary to the effect of an inert protein adsorbate. The distance separating the fluorophores is reduced, suggesting that this translocator provokes a perturbation in the palisade arrangement of lipid molecules in the bilayer.     

Modification of electroplax excitability by veratridine

Veratridine influences membrane-potential changes arising both from the action potential and from the application of external cholinergic agonists in the isolated monocellular electroplax preparation. The action potential shows a long depolarizing after-potential in the presence of veratridine. The effects of various pharmacological agents and of external ion changes on this after-potential are similar to those reported for other nerve and muscle fibers and are consistent with the view that veratridine acts chiefly to increase the Na⁺ conductance.Membrane depolarizations by cholinergic agonists are inhibited by veratridine at pH 7 but strikingly amplified at pH 9. The former effect appears to involve interaction with the cholinergic receptor at the surface of the membrane, while the latter potentiation parallels the increase in the spike after-potential at pH 9 and presumably arises from a Na⁺ conductance increase.Veratridine appears to interact with the component involved in the Na⁺ conductance in the interior membrane phase. The possible localization of this component in both the conducting and synaptic membrane is discussed.     

Lymphocyte-induced production of prostaglandin E2 by tumor cells in vitro: Requirements for direct contact and lymphocyte viability

The production of prostaglandin E₂ by tumor cell lines in response to exposure to purified lymphocytes has prompted the suggestion that this phenomenon may represent a defense mechanism whereby tumors may subvert an immune response mounted against them. To further characterize this phenomenon, cell lines derived from carcinogen-induced bladder tumors and embryo fibroblasts in Fischer rats were incubated with purified lymphocytes from peripheral blood, spleen, thymus, and lymph nodes from Fischer rats under a variety of conditions, and the amount of prostaglandin E₂ (PGE₂) production was determined by radioimmunoassay. Increased numbers of blood or splenic lymphocytes were associated with the induction of increased levels of PGE₂ production by the tumor cells. However, no prostaglandin was produced by the tumor cells after exposure to thymus or lymph node lymphocytes. Irradiation of lymphocytes prior to exposure to the tumor cells led to lower levels of PGE₂ production by the tumors, as did sonication of the lymphocyte preparations prior to addition to the tumor monolayers. Separation of lymphocytes from direct contact with the tumor cells resulted in less PGE₂ production by the tumor cell lines; however, when these lymphocytes were later layered onto fresh tumor cell monolayers, PGE₂ production occurred. Results in the present study suggest that direct contact between intact, viable, functionally active lymphocytes and tumor cells is necessary for tumor cell prostaglandin production to occur. Moreover, PGE₂ production only appears to occur in response to exposure to particular populations of lymphocytes, and this may correlate with the number of specific effector or attacker lymphocytes that are present. This specificity of response to effector cell challenge may be important in probing the defense mechanisms tumor cells may have to lymphocyte challenge, as well as in gauging the efficacy of a particular cellular immune response as it may be regulated both by cells involved in effecting this response as well as by the targets in lymphocyte/tumor cell interactions.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.