RNA synthesis in the follicular epithelium of the milkweed bug ovary

Sucrose density gradient analysis of the synthesis of ribosomal RNA during follicle cell maturation has revealed two distinct peaks of synthesis. The first of these is the largest and occurs during the transition from the delta to the gamma stages of oöcyte development. It is postulated that this increase in ribosomal synthesis is associated with both yolk uptake and subsequent growth of the follicular epithelium. A second, smaller, increase in the rate of synthesis occurs during the transition from the beta to the alpha₁ condition and this appears to be associated with the synthesis of the chorion by the follicular epithelium.     

Focus: Sex and Gender Health: Tobacco Smoking and the Resting Maternal Brain: A Preliminary Study of Frontal EEG

Tobacco smoking has been attributed to a wide range of detrimental health consequences for both women and their children. In addition to its known physical health effects, smoking may also impact maternal neural responses and subsequent caregiving behavior. To begin investigating this issue, we employed electroencephalography (EEG) to examine resting neural oscillations of tobacco-smoking mothers (n = 35) and non-smoking mothers (n = 35). We examined seven EEG frequency bands recorded from frontal electrode sites (delta, theta, alpha, alpha₁, alpha₂, beta, and gamma). While no between-group differences were present in high-frequency bands (alpha₂, beta, gamma), smokers showed greater spectral power in low-frequency bands (delta, theta, alpha, alpha₁) compared to non-smokers. This increased power in low-frequency bands of tobacco-smoking mothers is consistent with a less aroused state and may be one mechanism through which smoking might affect the maternal brain and caregiving behavior.     

Variation in the polysome assembly and incorporation of [3H]-uridine in the cells of pine buds during the cold season

The ribosome assemblies isolated from buds of Scots pine ( Pinus sylvestris L.) containing microsporangiate strobili varied both quantitatively and qualitatively in samples collected from October to April. The seasonal fluctuation in the amount of ribosonnes was more evident in the cytosolic fraction than in the smaller membrane-bound fraction. The profiles obtained after sucrose density gradient centrifugation were of two types. One type was commonly obtained from samples collected late in the autumn and early in the spring, and this type was characterized by a relatively high peak for the large subunits, a low or negligible peak for the dimers, and an even or ascending series of peaks for the polymers. The other type was obtained from samples collected during the winter, and was characterized by small peaks for both subunits, a moderate to large peak for the dimers and a descending series of peaks for the polymers. However, the scanning electron microscope investigations indicated that the winter-time samples did not lack polysomes and clusters of ribosomes. They did not become visible in the polysome profiles because they pelleted too tightly at the bottom of the centrifuge tubes to be removed with gradient fractionation. The au-toradiographic analyses suggested that the cells were capable of synthesizing mRNA throughout the winter, whereas rRNA synthesis was arrested. On the basis of the above results, we postulate that the synthesis of the enzyme proteins needed for the maintenance of winter-time metabolism takes place in the cytosolic ribosome fraction. The possible existence of winter-time polysome stores is also pointed out.     

Role of alpha 1 adrenoceptors in the turnover of phosphatidylinositol and of alpha 2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin?phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha₁ adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ?prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha₂ adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha₁ and alpha₂ adrenoceptors but about 90% of the binding sites were alpha₂. These data support the hypothesis that alpha₂ effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha₁ adrenoceptor activation.     

Alpha-adrenergic receptor subtypes: quantitative assessment by ligand binding.

We describe a method for quantitatively determining the alpha- adrenergic receptor subtypes in membrane fractions by studying the displacement of [³H] dihydroergocryptine by selective alpha antagonists and analyzing this data by a computer modeling technique. Alpha₁ receptors are those with a higher affinity for prazosin than for yohimbine; alpha₂ receptors have a higher affinity for yohimbine than for prazosin. Phentolamine does not discriminate between the two receptor subtypes present in rabbit uterus. The alpha receptor population of rabbit uterus was found to be 37% alpha₁ receptors and 63% alpha₂ receptors. The human platelet and rat liver alpha receptors were determined to be exclusively alpha₂ and alpha₁, respectively. In the uterus, prazosin had a 8800 fold greater affinity for alpha₁ than alpha₂ receptors while yohimbine had a 510 fold greater affinity for alpha₂ than alpha₁ receptors. The use of [³H] dihydroergocryptine displacement curves generated with selective alpha receptor antagonists coupled with subsequent computer modeling provides a precise and powerful method for quantifying the alpha receptor population of a tissue; this technique should be of value in studying the detailed regulation of alpha receptors in tissues which have both alpha₁ and alpha₂ receptors.     

Fat metabolism in higher plants. Differential incorporation of acyl-coenzymes A and acyl-acyl carrier proteins into plant microsomal lipids.

Coupling factor 1 and ribulose-diphosphate carboxylase are the main peripheral proteins associated with chloroplast internal membranes. The two proteins were sequentially solubilized and purified by gel filtration and their subunit structure was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The differences between the polypeptide profiles of the insoluble membrane fraction, before and after extraction of these oligomeric proteins, allowed identification of original membrane peptides with specific protein subunits. The 52,000 and 14,000 molecular weight peaks are identical to the large and small subunits, respectively, of ribulose-diphosphate carboxylase; the 56,000 and 53,000 peaks are identified with the α and β subunits, respectively, of the coupling factor protein. These identifications, together with earlier studies on the 25,000 M_r band, assign a physiological role to the most prominent peptides of chloroplast internal membranes. Now it becomes apparent that the major membrane polypeptides do not directly relate to photosynthetic electron transport components, but rather to enzymatic capacities associated with this process and to the light-gathering antenna of the photosynthetic unit. The observation that chloroplast coupling factor 1 dissociates during gel filtration, with preferential loss of the smaller subunits (M_r < 50,000) is discussed in relation to the possible function of these subunits in situ in the thylakoid membrane.     

Changes in the polysome content of developing Xenopus laevis embryos

A method for preparing polysomes from all embryonic stages of Xenopus laevis is described. In the oocyte only about 1–2% of the total ribosomes are present in polysomes, the remainder being a developmental reserve. Upon conversion to an egg the polysome content rises by up to 3-fold, and by about a further 2-fold after fertilization. There is only a small further increase during cleavage, but by the tailbud stage, when organogenesis begins, there is a more rapid rise. Most of the ribosomes are incorporated into polysomes by stage 42, shortly before feeding begins.At very early stages, the changes in polysome content seem to mirror the changes in protein synthesis. At later stages the polysome contents reported here provide the only available guide to changes in the rate of protein synthesis. Judged by polysome content, the stage 42 tadpole seems to make protein about 20 times faster than the unfertilized egg, though it contains very few more ribosomes. The relationship between polysome content and the synthesis of various types of RNA is discussed.     

Roles of Adrenergic α1 and Dopamine D1 and D2 Receptors in the Mediation of the Desynchronization Effects of Modafinil in a Mouse EEG Synchronization Model

Background

Synchronized electroencephalogram (EEG) activity is observed in pathological stages of cognitive impairment and epilepsy. Modafinil, known to increase the release of catecholamines, is a potent wake-promoting agent, and has shown some abilities to desynchronize EEG,but its receptor mechanisms by which modafinil induces desynchoronization remain to be elucidated. Here we used a pharmacological EEG synchronization model to investigate the involvement of adrenergic α₁ receptors (R, α₁R) and dopamine (DA) D₁ and D₂ receptors (D₁Rs and D₂Rs) on modafinil-induced desynchronization in mice.

Methodology/Principal Findings

Mice were treated with cholinergic receptor antagonist scopolamine and monoamine depletor reserpine to produce experimental EEG synchronization characterized by continuous large-amplitude synchronized activity, with prominent increased delta and decreased theta, alpha, and beta power density. The results showed that modafinil produced an EEG desynchronization in the model. This was characterized by a general decrease in amplitude of all the frequency bands between 0 and 20 Hz, a prominent reduction in delta power density, and an increase in theta power density. Adrenergic α₁R antagonist terazosin (1 mg/kg, i.p.) completely antagonized the EEG desynchronization effects of modafinil at 90 mg/kg. However, DA D₁R and D₂R blockers partially attenuated the effects of modafinil. The modafinil-induced decrease in the amplitudes of the delta, theta, alpha, and beta waves and in delta power density were completely abolished by pretreatment with a combination of the D₁R antagonist SCH 23390 (30 µg/kg) and the D₂R antagonist raclopride (2 mg/kg, i.p.).

Conclusions/Significance

These results suggest that modafinil-mediated desynchronization may be attributed to the activation of adrenergic α₁R, and dopaminergic D₁R and D₂R in a model of EEG synchronization.     

Polysome Formation in Light-sensitive Common Purslane Seeds

Common purslane (Portulaca oleracea L.) seeds show light-controlled dormancy. Ribosome profiles from dark-incubated seeds consist of 22 to 26% polysomes. Light induces germination and stimulates polysome formation during the 12-hour lag period preceding radicle protrusion. Polysome levels increase to 29, 35, and 41% with exposure to 3, 6, and 9 hours of light, respectively. Although polysomes form on imbibition in the dark, 6 hours of light stimulates a significant increase in polysome formation which is probably related to early stages of radicle elongation.     

Evidence for a novel circulating alpha 1 protein in tumour-bearing rats. Differentiation from acute phase alpha 1 proteins and from alpha 1 fetoprotein

It has been found in this study that the serum from rats bearing a transplanted dibenzanthracene-induced tumour ($\text{RD}\text{3}$), has a high concentration of alpha₁ proteins compared with normal rat serum. These alpha₁ proteins have been isolated by an immunoabsorption method and have been compared by immunological methods with the acute phase alpha₁ proteins isolated by the same method from the serum of rats presenting an inflammatory reaction. It has been found that the isolated $\text{RD}\text{3}$ alpha₁ proteins were composed of two major proteins: one of these corresponded to an inflammatory protein, the alpha₁-AP-globulin. The other may be a new protein, as it is absent from the serum of rats with an acute phase inflammatory reaction and nor does it correspond to alpha₁ feto-protein, a carcino-embryonic protein presenting the same electrophoretic mobility.     

Alpha-adrenergic receptors in liver membranes: delineation with subtype selective radioligands

Alpha₁ and alpha₂ adrenergic receptors have previously been demonstrated in rat liver membranes by competition curves of [³H]dihydroergocryptine ([³H]DHE) with the alpha₁ selective antagonist prazosin (B.B. Hoffman, D. Mullikin-Kilpatrick and R.J. Lefkowitz, J. Biol. Chem. $\text{255}$:4645–4652, 1980). The present studies have utilized the radioligands [³H]prazosin and [³H]yohimbine to further define alpha receptors in rat liver membranes. [³H]Prazosin was found to label alpha₁ receptors whereas [³H]yohimbine labelled alpha₂ receptors. The proportion of alpha₁ and alpha₂ receptors determined directly with these radioligands (79% and 20% respectively) was in good agreement with the proportions determined previously with [³H]DHE. Guanine nucleotides were found to reduce the affinity of the agonist epinephrine at the alpha₂ sites labelled by [³H]yohimbine but not at the alpha₁ sites labelled by [³H]prazosin. These results have implications for the interpretation of agonist interactions with alpha receptors in liver membranes.     

Role of phosphatidylinositol turnover in alpha1 and of adenylate cyclase inhibition in alpha2 effects of catecholamines

Ligand binding and pharmacological studies have indicated that alpha-adrenergic receptors can be divided into alpha₁ and alpha₂. We suggest that alpha₁ receptors mediate those metabolic effects of alpha catecholamines which involve phosphatidylinositol turnover and the release of bound intracellular Ca²⁺ as well as the entry of extracellular Ca²⁺. In contrast, alpha effects of catecholamines are due to non-specific inhibition of adenylate cyclase through a mechanism independent of Ca²⁺. A similar classification for the effects of both histamine and serotonin suggests that they have separate type 1 or alpha receptors for Ca²⁺ dynamics which are different from type 2 or beta receptors which regulate adenylate cyclase.There is a significant correlation between hormone effects on phosphatidylinositol turnover and elevation of intracellular Ca²⁺. The available data suggest that the turnover of membrane-bound phosphatidylinositol is involved in Ca²⁺ gating in rat hepatocytes, rat and hamster adipocytes and blowfly salivary glands. In hamster adipocytes adenylate cyclase activity is also inhibited by alpha₂ catecholamines through a Ca²⁺ independent mechanism.     

Separation and characterization of two bovine alpha1-fetoprotein molecular variants by concanavalin A-Sepharose chromatography.

Two molecular variants of bovine alpha₁-fetoprotein were separated by affinity chromatography of fetal calf serum on a concanavalin A-Sepharose column. Radialimmunodiffusion assay of bovine alpha₁-fetoprotein revealed that 29% of the alpha₁-fetoprotein in fetal serum lacked concanavalin A-binding activity whilst 71% of the alpha₁-fetoprotein was capable of binding to the lectin. These two bovine alpha₁-fetoprotein variants show antigenic identity suggesting that the polypeptide chain rather than the carbohydrate moiety of the alpha₁-fetoprotein molecule is the antigenic determinant.     

The action of exogenous gibberellic acid on polysome formation and translation of mRNA in germinating castor-bean seeds

The amount of protein synthesis in germinating castor-bean seeds has been estimated by the quantitative and qualitative exmainatin of polysomes from the seeds in the presence and absence of gibberellic acid (GA₃). Careful optimisation of polysome extraction procedures was required to minimise the ribonuclease activity in the extracts. Ribonuclease activity in seed extracts increased fourfold over the first 5 d of germination. Gibberellic acid stimulated polysome formation about twofold during the first 4 d of germination. It also stimulated the amount of mRNA associated with polysomes by about twofold during the first 3 d of germination. Between days 1 and 5 of germination, polysome formation was primarily limited by mRNA availability. During the period 0–24 h, polysome formation was independent of mRNA levles. The increase in enzyme activities stimulated by GA₃ was probably the result of an increase in the amount of cellular mRNA. No evidence was obtained for an action of GA₃ on translation other than on the increased production of RNA. Examination of the recruitment of isocitrate-lyase mRNA into polysomes showed that GA₃ did not specifically stimulate production of this enzyme.     

Dependence of the quality and the in vitro translation capacity of pine ribosome assemblies on the isolation procedure

The in vitro translation capacity of total ribosome assemblies isolated from the vegetative buds of small Scots pine (Pinus sylvestris L.) plants depends on the isolation procedure. Good yields and high values for protein synthesis were obtained in experiments in which polyvinyl pyrrolidone (PVP) was added to the grinding buffer. The polysome profiles obtained after sucrose density gradient centrifugation indicated the presence of polysomes in all samples. In addition, large ribosome aggregates were visible in the scanning electron micrographs. The use of an RNase inhibitor (RNasin) together with PVP did not improve the results, and treatment with ribonuclease (RNase, EC 3.1.27.5) destroyed the ability to synthesize protein. D, L-Dithiothreitol (DTT) and mercaptoethanol, if used instead of or together with PVP, gave low yields and also DTT destroyed the in vitro translation capacity of the ribosome assemblies. The polysome profiles had a high peak indicating dimers and often a descending series of peaks indicating polymers. A study of the scanning electron micrographs gave the impression that the largest polymers and aggregates had broken down. Protease K (EC 3.4.21.14) when added to the grinding buffer also destroyed the ability of the ribosomes to maintain protein synthesis in vitro. In this case, the shape of the polysome profiles gave the impression of successful isolation. Clumps of ribosomes, presumably originating from large aggregates, were visible in the scanning electron micrographs. Triton X-100 and 0.25 M NaCl in the grinding buffer extracted chromatin, which affected the results. The material lost during the extraction and purification processes consisted mainly of monosomes and their sub-units. On the basis of the above results it was concluded that the preservation of large polysomes and ribosome aggregates in the isolated ribosome assemblies is necessary if they are to maintain a high translation capacity. The content of the assemblies was best revealed in the scanning electron micrographs. The shape of the polysome profiles did not always correlate with the ability of the isolated ribosomes to synthesize proteins.     

Alpha2 adrenergic receptors are located prejunctionally in the Auerbach's plexus of the guinea pig small intestine: Direct demonstration by radioligand binding

We provide direct evidence that alpha₂-receptors in the guinea pig small intestine are localized prejunctionally in neurons of the Auerbach's plexus. The alpha₂-agonist ligand [³H]clonidine bound to a single saturable class of sites with a K_d of 1–2 nM and a capacity of approximately 70 fmol/mg protein in membranes from the innervated longitudinal and circular muscle layers of the intestine. By a special dissection technique the Auerbach's plexus could be completely removed from the longitudinal muscle. In these denervated preparations the clonidine binding sites were virtually completely removed whereas the expected binding was observed in innervated controls. The innervated preparations also contained a small number of alpha₁-receptors as revealed by binding with [³H]prazosin (capacity approximately 18 fmol/mg protein with a K_d of 0.4-0₃7 nM). Thus, the present study suggests that alpha₂-receptors ([³H]clonidine binding sites) are localized in neurons (i.e., prejunctionally) in the Auerbach's plexus of the guinea pig small intestine.     

Phorbol esters inhibit alpha1 adrenergic stimulation of glycogenolysis in isolated rat hepatocytes

Tumor promoting phorbol esters can stimulate Ca⁺⁺-phospholipid-dependent protein kinase. It has been suggested that this enzyme may mediate the effects of calcium-dependent hormones. In this paper the effects of phorbol 12-myristate 13-acetate (TPA) on isolated rat hepatocyte metabolism were studied. Phorbol esters completely blocked alpha₁-adrenergic stimulation of glycogenolysis. This effect is quite specific for alpha₁-adrenergic actions, as the stimulations of glycogenolysis by vasopressin, angiotensin II, ionophore A-23187 and glucagon were unaffected by TPA. The potencies of the different phorbol esters used in this study suggests that the inhibitory effects of these agents may be due to activation of protein kinase C. The effect of phorbol esters on alpha₁-adrenergic actions seems to occur at an early step of the alpha₁-adrenergic action. TPA (10^?11–10^?6M) was unable to stimulate glycogenolysis. Urea synthesis, which is stimulated by vasopressin and alpha₁-adrenergic agents, was not stimulated by phorbol ester, neither alone nor in combination with the Ca⁺⁺ ionophore A-23187.     

Comparative effects of ethylene and cyanide on respiration, polysome prevalence, and gene expression in carrot roots

Treatment of carrot roots (Daucus carota L.) with 10 microliters per liter ethylene in O₂ evokes a three- to four-fold increase in polysome prevalence and associated poly(A)⁺ RNA. The increase in polysome prevalence is attended by a similar change in CO₂ evolution. The increase in polysomal poly(A)⁺ mRNA constitutes primarily a generic increase in constitutive mRNAs as assayed by in vitro translation. However, changes in the relative abundance of several in vitro translatable ethylene specific mRNAs do occur.

Cyanide, at concentrations which inhibit cytochrome oxidase, initiates a respiratory rise very similar in kinetics and magnitude to that evoked by ethylene. Moreover, the combined treatment with cyanide and ethylene evokes a respiratory response resembling that caused by ethylene or cyanide alone. Nevertheless, cyanide, in the presence of ethylene, significantly inhibits the increase in polysome prevalence and new gene expression associated with ethylene treatment of carrot roots. Separation of in vitro translation products by one-dimensional and two-dimensional gel electrophoresis shows that several new in vitro translation products appear in cyanide-treated carrots different from those evoked by ethylene. Engagement of the less energy efficient alternative electron transport path by cyanide may be responsible for inhibition of the normal ethylene associated increase in polysome prevalence and new gene expression. The implications of these results on regulation of respiratory metabolism are discussed and compared with the results for similar experiments with avocado fruit (Tucker and Laties 1984 Plant Physiol 74: 307-315) in which cyanide does not inhibit an ethylene educed increase in polysome prevalence and change in gene expression.

     

Alpha and beta adrenergic receptors of canine lung tissue identification and characterization of alpha adrenergic receptors by two different ligands

Adrenergic receptors of canine peripheral lung tissues were measured by direct binding techniques using [³H]dihydroergocryptine ([³H]DHE), [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA). All three ligands bound to canine lung tissue with saturability, stereospecificity and reversibility. Adrenergic agonists competed for binding of [³H]DHE and [³H]prazosin in the order: 1-epinephrine > 1-norepinephrine > d-epinephrine > d-norepinephrine > 1-isoproterenol. Adrenergic antagonists competed for binding of [³H]prazosin in the order: prazosin > phentolamine > yohimbine. Inhibition curves of [³H]DHE by prazosin or yohimbine were biphasic suggesting two subtypes of binding sites. Maximum binding capacities of [³H]DHE ranged from 30.6 to 42.7 fmol/mg protein. [³H]prazosin from 18.3 to 26.9 fmol/mg protein and [³H]DHA from 135.2 to 359.4 fmol/mg protein. When both [³H]DHE and [³H]prazosin were used in the same membrane preparation, specific binding of [³H]DHE was always more than that of [³H]prazosin. Since [³H]prazosin is considered to bind to alpha₁ adrenergic receptors specifically and [³H]DHE is considered to bind alpha₂ adrenergic receptors nonselectively, the difference between the numbers of the specific binding sites of these two ligands should represent alpha₂ adrenergic receptors. Alpha₂ adrenergic receptor density ranged from 9.5 to 21.1 fmol/mg protein. Our results suggest the existence of both alpha₁ and alpha₂ adrenergic receptors in canine peripheral lung tissue. Approximately 40% of alpha adrenergic receptors were alpha₂. The ratio of alpha/beta adrenrgic receptors ranged from 1:3.3 to 1:10.4. The ratio of alpha₁/be ta adrenergic receptors ranged from 1:6.7 to 1:21.1.     

On the rate of ribosome translocation anisotropy and ribosome saturation in the polysome

This study examines the rate of ribosome translocation in the mammalian polysome engaged in protein synthesis by utilizing our knowledge of the hydrodynamic behavior of the rat liver polysomes, sedimenting in a linear sucrose density gradient. The average distance between adjacent ribosomes in the polysome was estimated assuming an extended linear configuration of the polysomes during sedimentation. Based on this estimate, the velocity of ribosome movement along the messenger RNA appears to be non-uniform and inversely related to the ribosome content of the polysome. Such non-uniformity prevails at stages of translation prior to ribosome “saturation” of the polysome. A correlation has been made between the results reported herein and previously published evidence on the rate of polypeptide chain synthesis. The steady-state condition for the polypeptide chain assembly is viewed as representing the state of ribosome “saturation”, characterized by a minimal ribosome velocity and a maximum density of ribosome distribution, both functions being uniform throughout the entire length of the polysome.