Gamma amino butyric acid (GABA) levels in hypophyseal stalk plasma of rats

To determine the possible physiologic contribution of GABA to the tonic hypothalamic inhibition of adenohypophyseal prolactin secretion, we compared GABA levels in hypophyseal stalk plasma with those found in the peripheral circulation. Hypophyseal stalk blood was collected $\text{via}$ a parapharyngeal approach from 8 urethane anesthetized diestrus rats. Peripheral blood was collected simultaneously from the external jugular vein of the same rat at a rate similar to hypophyseal stalk blood flow. Blood samples resulting from a single 4 hr collection per animal were centrifuged, and the plasma stored frozen before ethanol extraction and assay using a radioreceptor method. GABA levels in hypophyseal stalk plasma ($\text{909±171}\text{pmol/ml}\text{;}\text{X}\text{±}\text{S.E.M.}$) were not significantly higher than levels in peripheral plasma (845±182; p>0.05), indicating little or no secretion of GABA by the median eminence.     

In vitro and in vivo effects on brain GABA metabolism of (S)-4-amino-5-fluoropentanoic acid,a mechanism-based inactivator of γ-aminobutyric acid transaminase

The effects of intraperitoneal administration of (S)-4-amino-5-fluoropentanoic acid, a mechanism-based covalent inactivator of γ-aminobutyric acid transaminase (GABA-T), on whole brain GABA metabolism in mice were investigated. A dose-dependent and time-dependent irreversible inactivation of GABA-T was observed with a concomitant increase in whole brain GABA levels. The compound exhibited no $\text{in vitro}$ nor $\text{in vivo}$ time-dependent inhibition of glutamate decarboxylase (GAD), alanine transaminase, or aspartate transaminase (Asp-T). It was, however, a potent competitive reversible inhibitor of GAD and a weak competitive inhibitor of Asp-T. The chloro analogue, (S)-4-amino-5-chloropentanoic acid, was ineffective.     

Immunoassay for honey bee cytochrome c in single animals with cytochrome c-coated bacteriophages: A sensitive tool for the study of caste formation in the honey bee, Apis Mellifera

The development of a sensitive viroimmunoassay for honey bee cytochrome $\text{c}$ and its usage for early detection of caste differentiation is described. Pure honey bee cytochrome $\text{c}$ was isolated from workers and used to produce antibodies in rabbits. Bacteriophage T4 was chemically modified by covalent attachment of honey bee cytochrome $\text{c}$ using tolylene-2,4-diisocyanate as a cross-linking agent. The immunospecific inactivation of this bacteriophage-cytochrome c conjugate by anti-cytochrome $\text{c}$ antibodies can be inhibited by free cytochrome $\text{c}$. In quantitative determinations, 50% inhibition is reproducibly achieved at a concentration of 6 ng/ml (5 pmol/ml) and as little as 0.3 ng/ml (0.25 pmol/ml) could be detected by this system. Cytochrome $\text{c}$ concentrations were measured in individual animals and substantial differences between corresponding larval stages of worker and queen bees are reported.     

Evidence for central neuropeptidergic control of rumination in sheep

Effects of intracerebroventricular (ICV) vs. intravenous (IV) administration of tetragastrin, pentagastrin, CCK₈ and gastrin 17 on rumination were investigated in conscious sheep. Administered at 26 pmoles/kg ICV both tetra and pentagastrin induced a premature short (15–27 min) period of rumination only $\text{24±7}$ and $\text{23±9}$ min after food distribution in place of $\text{112±44}$ min in controls. Similar but less pronounced effects were observed for ICV administration of an equimolar dose of gastrin 17 whereas CCK₈ did not promote an early peciod of rumination despite its anorectic effects. Administered intravenously tetra and pentagastrin but not gastrin 17 caused early rumination only for 10 times higher doses. It is concluded that gastrin 17 and its C-terminal tetrapeptide may play a physiological role in the central control of rumination in sheep.     

The effect of stratification on in vitro protein synthesis in seeds of Pinus lambertiana

Incorporation of ¹⁴C-phenylalanine in $\text{in vitro}$ systems from sugar pine ($\text{Pinus lambertiana}$) seeds was studied. Embryo ribosomes from both dry and stratified seeds supported incorporation (431 and 326 pmoles, respectively, of phenylalanine per mg ribosome) when combined with an embryo pH 5 fraction from stratified seeds. Female gametophyte ribosomes from dry seeds were active (302 pmoles phenylalanine incorporated per mg ribosome) but lost 61 percent of their capacity to support protein synthesis after 35 hours' stratification. The pH 5 fraction from embryos increased in capacity to support incorporation as stratification progressed up to 60 days (398 pmoles phenylalanine per mg ribosome when ribosomes were from 90-day stratified embryos) while the pH 5 fraction from female gametophytes was never active.     

Phosphorylated guanosine derivatives of eukaryotes: Regulation of DNA-dependent RNA polymerases I,II, and III in fungal development

Three phosphorylated guanosine derivatives designated HS-1, HS-2 and HS-3 synthesised during active protein synthesis in the water-mould, $\text{Achlya}$ sp (1969) were shown to regulate the enzymatic activities of nucleoplasmic and nucleolar DNA-dependent RNA polymerases (RNAP-I, II and III) from both $\text{Achlya}$ and another unrelated water-mould, $\text{Blastocladiella emersonii}$. These HS compounds were without effect on $\text{E. coli}$ DNA-dependent RNA polymerase holoenzyme. The most potent of the three compounds was HS-3 which inhibited the activity of all enzymes completely at 100 μg/ml. HS-1, on the other hand, activated maximally at 1 to 10 μg/ml. HS-1 activation (3-fold) was restricted to enzyme III, and it had only partial inhibitory effects on enzymes I and II. The pattern of synthesis of HS-compounds throughout the 20-hour asexual growth cycle of the organism correlated with the detectable levels of the different RNA polymerases of $\text{Achlya}$.     

Induction and inhibition of meiotic maturation of follicle-enclosed porcine oocytes in vitro

Isolated porcine Graafian follicles which were explanted in vitro and maintained in organ culture were used as a test-system for the meiosis-inducing action of PMSG and hCG. The addition of either PMSG or hCG alone (10 or 20 IU/ml, respectively) to the culture medium was not effective, whereas the simultaneous administration of these hormones ($\text{15}\text{15}\text{IU/ml}$) induced resumption of meiosis in 90.3% ($\text{37}\text{41}$). The same hormone concentrations were used in a second series of experiments in which the inhibition and induction of meiosis of isolated oocytes were tested by transferring them into host follicles. In host follicles containing up to 12 foreign eggs, which were cultured in control media, meiosis was prevented in 86.0% of all oocytes ($\text{104}\text{121}$). By adding PMSG (15 IU/ml) simultaneously with hCG (15 IU/ml) to the medium, meiosis was induced in 95.0% of all oocytes ($\text{133}\text{140}$).The assumption is made that the signal initiating resumption of meiosis of the isolated and transferred oocytes is mediated by the follicular fluid, since intimate contact with the membrana granulosa of the host follicle was prevented by using a roller technique.     

Circulating immunoreactivities of gastrin,glucagon and VIP after jejuno-ileal bypass

Seven Sprague-Dawley rats (404–440 g) underwent a 90% jejuno-ileal bypass (JIB); the functional loop consisted of $\text{1}\text{3}$ ileum and $\text{2}\text{3}$ jejunum with the bypassed loop being anastamosed to the ascending colon. Seven control rats were sham-operated. After 35 days, the rats were fasted 18 hours and venous blood was collected. Immunoreactivity of gastrin, measured with an antibody binding equally to G17 and G34, was higher in the plasma of the JIB (256±55 SEM pg/ml) than control (85±9 pg/ml) rats. This agrees with recent human studies but is in conflict with results in less mature rats. VIP levels were not significantly different. Glucagon-like immunoreactivity measured with antibodies specific for the C- and N-terminal regions of the hormone, respectively, were also higher in the JIB (510±40 and 129±15 pg/ml) rats.     

Cyclic AMP levels in Drosophila during postembryonic development and in the adults.

The physiological content of Drosophila melanogaster tissues in cyclic AMP has been determined and its variations studied during postembryonic development and in the adults. Marked variations were observed, especially during metamorphosis where the ratio between the lowest and highest values (0·35 to 17·25 pmoles/mg protein) was $\text{1}\text{44}$. In larvae the variations of cyclic AMP level were not clearly related to the larval ecdyses, but the steps of metamorphosis, i.e. formation of the puparium, larval-pupal apolysis, and pupal-adult apolysis, were accompanied with rapid and drastic rises of cyclic AMP, up to the highest value mentioned. We therefore deduce that cyclic AMP is involved in the metamorphosis of D. melanogaster as a chemical signal. In adults, the cyclic AMP level was remarkably constant and was around 7 pmoles/mg protein.     

Plasma relaxin concentrations in the pig during the periparturient period: Association with prolactin,estrogen and progesterone concentrations

Concentrations of relaxin, prolactin, unchromatographed estradiol 17_β (E_2β) and progesterone (P₄) were measured in serial samples of inferior vena caval blood, in three pigs, during late pregnancy, and parturition. Maximal relaxin concentrations occurred 60 to 24h before parturition, and ranged from 60 to 286ng/ml. Prolactin concentrations increased from 12.5ng/ml, 48 to 36 hours before parturition, to between 79 to 184ng/ml. At the time of the relaxin surge, E_2β levels were high, and P₄ concentrations were decreasing, thus raising the $\text{E}{}_{\mathrm{2\beta }}\text{P}{}_4$ ratio. A surge in prolactin concentrations followed upon a decline of P₄ to less than 10ng/ml, coinciding with the increase in relaxin concentrations in 2 gilts, and following the surge in relaxin in the third. Udder development occurred near the time of increased relaxin concentrations. ‘Milk let down’ followed maximal relaxin and prolactin concentrations in two gilts, and the increase in prolactin, rather than the increase in relaxin concentration, in the third.     

Ethanol and other CNS depressants decrease GABA synthesis in mouse cerebral cortex and cerebellum in vivo

GABA synthesis in mouse brain $\text{in vivo}$ was estimated by measuring the rate of GABA accumulation one hour after inhibition of GABA degradation using the selective and irreversible antagonism of GABA-transaminase by gabaculine. Using this method we found that acute and repeated ethanol administration lead to a potent depression of gabaculine induced enhancement of GABA levels in mouse brain cerebellum and cerebral cortex. Alcohol, in the absence of gabaculine had no effect on steady state GABA levels. These results demonstrate potent effects of ethanol on the dynamics of GABA metabolism which are compatible with a GABA like effect of ethanol.     

3H-Benzene metabolism in rabbit bone marrow

An assay for benzene metabolism using ³H-benzene and high pressure liquid chromatography was developed. ³H-Benzene metabolism (2 pmoles benzene equivalents/mg protein/min) required the presence of a TPNH generating system and was inhibited 80% in the presence of a CO:O₂ (9:1) atmosphere. The products of ³H-benzene rabbit bone marrow microsomal metabolism were phenol and an unidentified metabolite. Cytochrome P-450 (26–51 pmoles/mg microsomal protein) and cytochrome $\text{c}$ reductase activity (7.8–21.0 nmole/mg microsomal protein/min) were detected in rabbit bone marrow.     

Response of two cows with mummified fetus to treatment with cloprostenol (ICI 80, 996)

Two cases of bovine fetal mummification were treated with a single intramuscular injection of 500 μg cloprostenol. Plasma progesterone levels fell rapidly within 24 hours following injection from a mean level of 10.45 ng/ml to 1.05 ng/ml. Extradiol-17β levels were variable and low ($\text{x}\text{= 3.04 pg/ml}$) during the 120-hour postinjection sampling period. The mummies were delivered at 104 and 120 hours postinjection. Both were expelled into the vagina and traction was required to complete removal from the genital tract. No adverse reactions which could be attributed to $\text{cloprostenol}$ treatment were observed.     

Semiautomated enzymic micro methods for blood glucose and lactic acid on a single filtrate

Methods have been modified and adapted to permit enzymic automated micro analysis of glucose on 0.021 ml of blood, and of lactic acid on 0.0115 ml of blood. By means of a single 1:20 Somogyi filtrate with a final volume of 1–1.4 ml prepared from 0.1–0.15 ml blood, both determinations can be accomplished individually on the autoanalyzer.The range of concentrations of each method has been extended so that glucose may be determined between 5 and $\text{200 mg}\text{100 ml}$ (or higher depending on dilution) and lactic acid between 5 and $\text{125 mg}\text{100 ml}$.The methods are sensitive, reproducible, and stable. The recoveries and the comparison studies with manual methods are well within the limits of clinical investigation procdures.     

Antinociceptive and hypothermic effects of trimethyltin

Trimethyltin (TMT) induced a dose-dependent antinociceptive and hypothermic effect in mice. Antinociception was not attenuated by naloxone but was reversed by atropine. TMT, however, was ineffective in displacing (³H)-QNB binding $\text{in vitro}$ and did not affect (³H)-QNB binding or acetylcholinesterase activity after $\text{in vivo}$ administration. The ethyl ester of nipecotic acid, a specific inhibitor of synaptosomal GABA uptake, exerted a similar antinociceptive effect that could be blocked by atropine. The GABA receptor antagonist bicuculline attenuated antinociception induced by TMT and nipecotic acid ethyl ester but not by morphine or oxotremorine. γ-Vinyl GABA, an irreversible inhibitor of GABA metabolism, prolonged TMT but not morphine-induced antinociception. In contrast, neither the dose-response nor the time course of TMT-induced hypothermia were affected by any of the drugs tested. The findings suggest that the GABAergic system may be involved in TMT induced antinociception; however, the mechanism responsible for the hypothermic effect of TMT is not apparent.     

Adenylate cyclase and cyclic AMP through the cell cycle of Tetrahymena.

Adenylate cyclase activity and 3′, 5′ cyclic adenosinemonophosphate (cAMP) have been followed through the heat-synchronized cell cycle of Tetrahymena pyriformis. While the specific activity of adenylate cyclase remained essentially constant throughout the cycle, cAMP oscillated (between 10 and 50 pmoles/mg protein) through two cycles. Minima were observed at each division ($\text{D}\text{S}$ border) and maxima at each $\text{S}\text{G}\text{2}$ border. Each heat shock caused slight temporary reduction in cyclase activity. Further observations suggest to us that adenylate cyclase shows conformational changes in response to temperature-induced alterations and to changes in lipid composition of membranes.     

Receptor-like stereospecific binding of a pyrethroid insecticide to mouse brain membranes

A heterogeneous particulate fraction of mouse brain homogenates binds NRDC 157 (3-phenoxybenzyl [1$\text{R}$,$\text{cis}$]-3-(2,2-dibromovinyl)-2,2- dimethylcyclopropanecarboxylate), a potent pyrethroid insecticide, stereospecifically and with high affinity. Stereospecific binding is a minor component of total binding (2.8%); the remainder of observed binding is predominantly nonspecific and unsaturable. Stereospecific binding is half-saturated at 4×10^?8$\text{M}$ and fully saturated at concentrations in excess of 1×10^?7$\text{M}$. The stereospecific binding capacity of this preparation was 200–250 pmoles of NRDC 157 per gram equivalent of brain tissue (2.3–2.8 pmol/mg protein). This binding site may represent the neural receptor involved in the stereospecific toxic action of pyrethroids.     

The pharmacokinetics of tritiated ethinylestradiol in the rabbit

The disappearance of ethinylestradiol from the blood of rabbits has been studied, following the intravenous administration of this steroid. The disappearance followed two exponentials, the first having a half life ($\text{t}\text{1}\text{2}$) of 5.5 min and the second, apparently terminal exponential was also rapid ($\text{t}\text{1}\text{2}\text{= 69}\text{min}$). The plasma clearance was 150 ml/min which suggests almost total clearance of this steroid during a single passage through the liver. Bile contained a significant concentration of EE conjugates and thus this steroid could undergo enterohepatic recirculations. A large oral dose of unlabelled EE, given prior to intravenous administra tion of tritiated EE, considerably altered the pharmacokinetics of the latter by saturating both phase one metabolism (changes of the steroid nucleus) and the secretion of conjugates into bile. It was not clear whether phase two metabolism (conjugation) was also saturated.     

Interactions of lindane,toxaphene and cyclodienes with brain-specific t-butylbicyclophosphorothionate receptor

Three major classes of chlorinated hydrocarbon insecticides, $\text{i}$. $\text{e}$., the lindane/hexachlorocyclohexane, toxaphene and aldrin/dieldrin types, are potent, competitive, and stereospecific inhibitors of $\text{t}$-butylbicyclophosphorothionate (TBPS) binding to brain-specific sites, thereby indicating an action at the γ-aminobutyric acid (GABA)-regulated chloride channel. The most inhibitory and toxic of four isomers of hexachlorocyclohexane is lindane and of >188 components of toxaphene is 2,2,5-$\text{endo}$, 6-$\text{exo}$, 8,9,9,10-octachlorobornane. 12-Ketoendrin (IC₅₀ = 36 nM) is twice as active as the most potent previously known inhibitor of TBPS binding and it is also the most inhibitory and toxic of 22 cyclodienes examined. Within each of these three series of polychlorocycloalkanes the mammalian toxicity is closely related to the potency for inhibition of TBPS binding. A modified receptor assay incorporating liver microsomes and reduced nicotinamide-adenine dinucleotide phosphate compensates in part for oxidative detoxification and bioactivation. Specific TBPS binding is reduced in a dose-dependent manner in dieldrin-poisoned rats. DDT, mirex and kepone are not inhibitors of TBPS binding, even at 10 μM.