Comparison of Solubilised Kainate and α-Amino-3-Hydroxy-5-Methylisoxazolepropionate Binding Sites in Chick Cerebellum

Abstract: [³H]Kainate bound to chick cerebellar membranes with a K _D of 0.6 μ M and with an exceptionally high B max of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [³H]kainate was reduced ( K _D= 2.7 μ M ), but the B _max was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate > kainate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > glutamate. Binding sites for α-[³H]amino-3-hydroxy-5-methylisoxazolepropionate ([³H]AMPA) were much less abundant, with K _D and B _max values in membranes of 86 n M and I pmol/mg of protein, respectively. The affinity of [³H]AMPA binding was also reduced on solubilisation ( K _D= 465 n M ), but there was an increase in the B _max (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [³H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [³H]kainate, domoate was markedly less potent than kainate at displacing [³H]AMPA. These results suggest that [³H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [³H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non- N -methyl-D-aspartate receptors.     

EVIDENCE FOR THE SYNTHESIS AND STORAGE OF 5-HYDROXYTRYPTAMINE IN TWO SEPARATE POOLS IN THE BRAIN

Abstract— Evidence is presented to support the hypothesis that 5-hydroxytryptamine (5-HT) in the rat brain is synthesized by two separate pathways and stored in two or more compartments. Lysergic acid diethylamide in doses down to 50 μg/kg was shown to reduce the formation of 5-[³H]HT from [³H]tryptophan in the presence of a monoamine oxidase inhibitor, although the total rate of accumulation of 5-HT was unchanged. Conversely, adrenalectomy was found to increase the total synthesis of 5-HT measured in the same way, although the amount of 5-[³H]HT formed suggested that there was no increase in the synthesis of the amine. In a third experiment it was found that electrical stimulation of 5-HT-containing nerves following labelling of 5-HT stores with [³H]tryptophan led to a biphasic disappearance of 5-[³H]HT. It is suggested that the method of measuring 5-HT synthesis by measuring 5-[³H]HT formed from[³H]tryptophan in the presence of a monoamine oxidase inhibitor may be a way of selectively measuring the turnover of the functional pool of 5-HT.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

Transport of G AB A, β-Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high-affinity carrier-mediated transport systems for [³H]GABA ( K _t=1.9 μM, V = 1.8 pmol/10⁶ cells/min) and [³H]glutamate ( K _t= 10 μM, V = 7.9 pmol/10⁶ cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [³H]β-alanine (which is taken up specifically by the glial GABA transport system) and [³H]glutamate were concentrated by glial-enriched fractions. However [³H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high-affinity [³H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high-affinity glutamate transport is a property of glial cells but not granule neurons.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

Determination of xanthoxin by immunoassay

2- Cis (-)xanthoxin (XA) was linked to bovine serum albumin through a Schiff's base and the adduct stabilized by sodium borohydride reduction. The conjugate (molar coupling ratio: 3 mol XA per mol protein) was highly immunogenic in rabbits. Antisera contained antibodies binding XA with high affinity (K_a= 1.8 × 10⁸ M ⁻¹). [³H]-XA (2.2 × 10¹⁴ Bq mol⁻¹) was synthesized by oxidation of [³H]-XA alcohol with MnO₂ and used to set up a radioimmunoassay [RIA, detection limit, 1 pmol; measuring range, 1 to 200 pmol (0.3 to 60 ng) XA]. The sera were also suitable for enzyme-linked-immunosorbent assay (ELISA) using XA-alkaline phosphatase conjugates. The technique was more sensitive [detection limit, 0.1 pmol; measuring range, 0.1 to 50 pmol (0.05 to 15 ng) XA] than the radioimmunoassay, but less precise.     

Changes of Nucleic Acids and Proteins in the Oocysts of Eimeria tenella during Sporulation

SYNOPSIS. The total content of DNA in Eimeria tenella , estimated at 5.8 × 10⁻¹² gm/oocyst, varies little during sporulation. Its buoyant density is 1.682 gm/cm³, reflecting a G + C content of ∼41%. Thymidine is not incorporated into any TCA insoluble fraction of sporulating oocysts, but radioactivity from [³H]uridine and [³H]deoxyuridine are incorporated into RNA at a linear rate during the first 5 hr of sporulation. The labeled RNA, found mainly in the paranuclear bodies of newly formed sporozoites, contains ∼0.15 nmole [³H]uridine/10⁶ oocysts at the completion of sporulation. One nmole of leucine is incorporated into the hot TCA insoluble fraction of 10⁶ oocysts during the first 7 hr of sporulation after an initial lag. The incorporated amino acid is mainly in the cytoplasm of the sporozoites, and an analysis by SDS-gel electrophoresis reveals most of the radioactivity in a narrow band with a molecular weight of ∼50,000 daltons. Incorporation of uridine and leucine, however, can be totally suppressed by respiratory inhibition. Further analysis of the proteins in the oocysts reveals that the total protein content remains relatively unchanged at 2.64 × 10⁻¹⁶ gm/oocyst during sporulation, but there is a shift of 13–14% of total protein from the soluble cytoplasm to the 15,000 g pellets. By polyacrylamide gel electrophoresis, a major protein band. possibly a glycoprotein, is shown in the soluble cytoplasm of unsporulated oocysts. This band disappears during sporulation.     

EFFECT OF PENTOBARBITAL ON MITOCHONDRIAL SYNTHESIS IN CULTURED GLIAL CELLS

Abstract— Primary cultures of glial cells prepared from brains of newborn rats were grown for 1 week and then exposed to 5 × 10⁻⁴ m -pentobarbital (PB) for 4 weeks. Compared with glial cells grown in drug-free medium throughout, exposure to PB significantly increased hexokinase activity, primarily in its mitochondrial form. Furthermore, cellular protein and RNA concentrations were significantly higher in barbiturate-cultivated than in control cells. Pulse labelling with [³H]thymidine after 4 weeks of PB exposure resulted in a significant increase (332%) in ³H incorporation into mitochondrial DNA while ³H incorporation into nuclear DNA was reduced by 58%. In addition, there was a time dependent increase in the size and number of mitochondria as determined in electron micrographs. These results are interpreted to reflect an increased mitochondrial metabolism in glial cells after chronic exposure to the barbiturate and may constitute a compensatory mechanism to the depressant action of this drug.     

Influence of the growth rate on the macromolecular composition of Acinetobacter calcoaceticus in carbon-limited chemostat culture

Abstract Infectious phage particles can be formed in vitro when extracts of T1-infected cells are incubated with T1 DNA. The DNA packaging system is based on mixtures of complementing extracts from Escherichia coli sup⁰ cells infected with the amber mutants am 4 (gene 16) or am 10 (gene 13). Gene 16 mutants are defective in the formation of DNA-filled heads but make proheads; gene 13 mutants are defective in prohead formation. Three forms of DNA have been packaged: (1) endogenous concatemeric DNA present in mixtures of am 4 and am 10 mutant extracts; (2) concatemeric DNA; (3) virion DNA both when supplied exogenously to mixtures of am 4 · am 20 and am 10 · am 20 double mutant extracts ( am 20 inhibits T1 DNA synthesis). The reaction requires added ATP, Mg²⁺ and spermidine for optimum efficiency and produces about 1.5 × 10³ pfu/ μ g and about 1 × 10⁴ pfu/ μ g for exogenous concatemeric and virion DNA, respectively.     

Hormone-Induced Alterations in Nonhistone Protein

Methylation and phosphorylation of chromosomal nonhistone protein (NHP) has been demonstrated in the salivary gland cells of diptera [5, 7] and implicated in the control of gene expression [35, 36]. Furthermore, hormones can stimulate methyl and phosphoryl side chain metabolism and thus enhance template activity. Salivary glands from late fourth instar female larvae of Sciara coprophila (cortisone-supplemented and normal diet) were incubated in ³H-uridine (10 μCi/ml), ³H-thymidine (10 μCi/ml), ³H-methyl-methionine (20 μCi/ml), ³⁵S-methionine (10 μCi/ml) and ³²P-orthophosphate (1 mc/ml), for varying time periods, to measure RNA synthesis, DNA synthesis, methylation, protein synthesis and phosphorylation, respectively. Following selective extraction of lipid, histone and nucleic acids, glands were prepared for light microscope autoradiography. A more specific labelling pattern, as well as increased grain number on particular bands, interbands and bulbs, was noted on chromosomes from cortisone-fed larvae incubated in ³H-methyl-methionine for 1 min when compared with larvae on the standard diet. Cortisone also increased RNA synthesis and nucleoprotein phosphorylation, but not DNA or protein synthesis. In summary, cortisone enhances the specificity and degree of NHP methylation and phosphorylation at discrete chromosomal loci, i.e. alterations in side chain metabolism which may be responsible for increased RNA synthesis.     

Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin

Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [³H]fucose or [¹⁴C]glycine precursors. The nerve slice system gave nearly linear incorporation of [³H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [³H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10⁻⁸ m to 1.5 × 10⁻⁶ m . Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P₀, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P₀ into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P₀ myelin.     

Uptake of [3H]Dopamine into Dopaminergic and Noradrenergic Neurones of the Isolated Neurointermediate Lobe of the Rat Hypophysis. Effects of Desipramine and Nomifensine

Abstract: The isolated neurointermediate lobe (NIL) of the rat hypophysis accumulates [³H]dopamine from the incubation medium. Column chromatographic analysis showed that 92% of the tissue radioactivity was contained in the catecholamine fraction. [³H]Dopamine represented 70% and [³H]noradrenaline 30% of the [³H]catecholamines. Desipramine (1 μM) prevented the formation of [³H]noradrenaline without affecting the storage of [³H]dopamine. Nomifensine (10 μM) blocked the storage of [³H]dopamine and [³H]noradrenaline. Thus, in the NIL, [³H]dopamine is taken up into dopaminergic and noradrenergic neurones. In the latter, [³H]dopamine is converted to [³H]noradrenaline, indicating a significant dopamine β-hydroxylase activity in the NIL tissue. A selective labeling of the dopamine stores with [³H]dopamine can be achieved in the presence of desipramine.     

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (−)nicotine on the function of the NMDA-mediated modulation of [³H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [³H]DA release in rats chronically treated with vehicle (14 days) with an EC₅₀ of 13.1 ± 2.0 μM. The NMDA-evoked overflow of the [³H]DA in PFC nerve endings of rats treated with (−)nicotine was significantly lower (−43%) than in vehicle treated rats. The EC₅₀ was 9.0 ± 1.4 μM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [³H]DA overflow with an EC₅₀ of 14.5 ± 5.5 μM. This effect was significantly enhanced in chronically treated animals. The EC₅₀ was 10.5 ± 0.5 μM. The K⁺-evoked release of [³H]DA was not modified by the (−)nicotine administration. Both the changes of the NMDA-evoked [³H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (−)nicotine differentially affects the NMDA-mediated [³H]DA release in the PFC and NAc of the rat.     

THE SELECTIVE NEURONAL UPTAKE AND RELEASE OF [3H]DL-2,4-DIAMINOBUTYRIC ACID BY RAT CEREBRAL CORTEX

Abstract— At 25°C the accumulation of [³H] dl -2,4-diaminobutyric acid (DABA) into small rat cortical slices was linear with time and a tissue: medium ratio of 35:1 was attained after 60 min. At 37°C the uptake was no longer linear and the tissue: medium ratio at 60 min was 66:1. Uptake was unaffected by the addition of 10 μ m -AOAA and dependent on the presence of Na⁺ in the incubation media. The uptake was shown to have a high affinity component with a K _m of 20.7 μ m and a V _max of 28.6 nmol/g/min. IC50's for the inhibition of [³H]DABA uptake by dl -DABA, l -DABA and GABA were 80, 40 and 17 μ m respectively. Two m m β -alanine, however, caused less than 13% inhibition of [³H]DABA uptake. Electron microscopic autoradiographs showed the [³H]DABA to be accumulated by 22% of the identifiable nerve terminals and, after 14 days exposure, the density of silver grains over nerve terminals was 36–38 times higher than that over the rest of the electron micrograph. On the other hand, [³H]DABA was not taken up into rat sensory ganglia and light level autoradiography showed the small amount of [³H]DABA accumulated by the ganglia to be evenly distributed throughout the tissue. Both electrical stimulation for 30 s and exposure of the tissue to a medium containing 47 m m -K⁺ for 2 min caused a marked increase in the efflux of [³H]DABA from the tissue. Both these effects were abolished by a reduction in Ca²⁺ concentration and an increase in the Mg²⁺ concentration of the superfusing medium. These results suggest that l -DABA acts as a 'false transmitter' for the neuronal uptake, storage and release of GABA.     

Acinar cell proliferation in the parotid and submandibular salivary glands of the neonatal rat

Abstract. Although the rat salivary glands are deficient in acini at birth, acinar cells proliferate rapidly during the early post-natal period. The pattern of acinar cell proliferation was analysed in the parotid and submandibular glands of neonatal rats from day of birth until day 34. Mitotic and [³H]thymidine ([³H]TdR) labelling indices of the two glands show distinctly different patterns. Analysis of cell division in the rat parotid gland demonstrated a peak of mitotic index at 14 days (2.9 ± 0.4%) and labelling index at 16 days (25.2 ± 2.1%). Submandibular gland acinar cell proliferation reaches a zenith between 7–8 days; labelling index (14.2 ± 1.1%) and mitotic index (2.3 ± 0.3%). Cell proliferation decreases rapidly in both glands after reaching a peak in activity. Gland size increases more rapidly in the submandibular gland which correlates with the earlier shift from cell proliferation to differentiation which occurs in this organ. Circadian rhythms of [³H]TdR incorporation were also investigated in this study. A circadian rhythm of [³H]TdR incorporation into DNA occurs at 15 days after birth with a peak at 06.00 hours in both glands and a trough occurring at 15.00 hours in parotid gland and 18.00 hours in the submandibular gland. Determination of specific activity of DNA (ct/min per μg DNA) on days 8, 10, 12, 13, 14, 15, and 16 after birth at 06.00 and 15.00 hours indicated that a circadian rhythm in [³H]TdR incorporation into DNA began on day 14. The developmental switch from suckling to solid food may be an initiating factor in the sychronization of the circadian rhythm in cell proliferation.     

Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions

Abstract: Guanine nucleotides differentiate binding of tritium-labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [³H]-labeled agonists by 20–60% and [³H]-labeled antagonists by 0–20%. In the presence of 100 mM-NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high-affinity [³H]dihydromorphine sites by 52% and low-affinity sites by 55%. Without sodium, GTP reduced high-affinity [³H]-naloxone sites by 36%; in the presence of 100 mM-NaCl, GTP had no effect on either high- or low-affinity [³H]naloxone sites. GTP increased the association rate of [³H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [³H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [³H]-diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40- 120-fold, with met- and leu-enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [³H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [³H]-labeled agonist binding by stimulating membrane-bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors.     

Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

Neurochemical Studies of the Mesolimbic Dopaminergic Pathway: Somatodendritic Mechanisms and GABAergic Neurones in the Rat Ventral Tegmentum

Abstract: The rat ventral tegmentum (containing dendrites and somata of mesolimbic neurones) contained 1.3 μg/g of dopamine, which was reduced to 40% of the control level by reserpine. Slices of ventral tegmentum were able to accumulate and release (elevated potassium or protoveratrine A) exogenous [³H]dopamine. In parallel studies the uptake mechanism in ventral tegmentum was shown to be virtually identical to the nerve terminal uptake of [³H]dopamine by slices of nucleus accumbens. The release of [³H]dopamine was indistinguishable from that observed in substantia nigra, where there is substantial evidence for dendritic mechanisms. Basal adenylate cyclase activity was present, but dopamine-stimulated activity was not detected. A high GABA concentration (7.7 μmol/g) was present in ventral tegmentum, in conjunction with an uptake and a release mechanism for [³H]GABA. GABA and muscimol elicited a small, reproducible efflux of [³H]dopamine, but an interaction between dopamine and [³H]GABA efflux was not observed. The results are in accord with transmitter roles for dopamine and GABA in the somatoden-dritic area of mesolimbic dopaminergic neurons.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.