Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

The binding of [³⁵S]GTPS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 M. The specific binding was 98%. Different guanine nucleotides were all able to compete with [³⁵S]GTPS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [³⁵S]GTPS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [³⁵S]GTPS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPS, did not displace [³⁵S]GTPS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [³⁵S]GTPS binding was strongly Mg²⁺-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [³⁵S]GTPS in the presence of GTP may reflect functional G-protein activity.     

Opioid and Cannabinoid Receptors Share a Common Pool of GTP-Binding Proteins in Cotransfected Cells, But Not in Cells Which Endogenously Coexpress the Receptors

1. Opioid (, , ) and cannabinoid (CB1, CB2) receptors are coupled mainly toG_i/G_o GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins.2. The stimulation of [³⁵S]GTPS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of - and -opioid receptors led to stimulation of [³⁵S]GTPS binding by either -selective (DAMGO) or -selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes.3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either - or -opioid receptors.4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and -opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress - and -opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptorsubtype.5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.     

Detection of Cannabinoid CB1, Adenosine A1, Muscarinic Acetylcholine, and GABAB Receptor-Dependent G Protein Activity in Transducin-Deactivated Membranes and Autoradiography Sections of Rat Retina

1. Several G-protein-coupled receptors (GPCRs) have been localized to various layers of the vertebrate retina, using autoradiographic and immunohistochemical techniques, but the functional data concerning G protein activation are limited. Here, we establish optimized assay conditions to detect receptor-dependent G protein activity in membranes and tissue sections of the rat retina. 2. Agonist-stimulated [35S]GTPgammaS-binding responses were characterized for the Gi/o-linked adenosine A1, cannabinoid CB1, m2/m4 muscarinic acetylcholine, and GABA(B) receptors. Initial assumption was that G protein activity under "basal conditions" is high due to enrichment and activity of rhodopsin and transducin in this tissue. 3. We found that pretreatment of retina membranes with hydroxylamine (10 mM), a rhodopsin-inactivating drug, substantially (up to 60%) reduced basal G protein activity, thereby improving signal-to-noise ratio to detect agonist-stimulated G protein activation for all studied receptors. [35S]GTPgammaS autoradiography revealed that hydroxylamine specifically reduced basal binding in the transducin-enriched photoreceptor layer. In contrast, hydroxylamine did not affect GPCR signaling in brain membranes, indicating specific action on retinal transducin. 4. For all studied receptors, [35S]GTPgammaS autoradiography allowed localization of G protein activity to different retinal layers, with the bulk of signal detected in the ganglion cell layer. Strongest responses were observed for adenosine and muscarinic receptor agonists. Additional G protein activity was detected in the inner plexiform layer. 5. Responses to all tested agonists were reversed in the presence of appropriate receptor-selective antagonists, indicating receptor-mediated G protein activation.     

Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation

Myristoylated G_o was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G_o) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_o, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5"-3-O-(thio)triphosphate (GTPS) activation induced disaggregation of the G_o oligomers to monomers. The G_o was crosslinked by a cross-linker, N,N"-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G_o was pretreated with GTPS. Immunoblot analysis also demonstrated oligomerization of the purified G_o proteins and its disaggregation triggered by GTPS. These results provided direct evidence for the disaggregation–coupling theory and the disaggregation action of GTPS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.     

Studies on the iodination of aras protein and the detection ofras polymers

Several methods for the iodination of recombinant v-H-ras protein were compared. The Iodobead method gave greates incorporation of radioactivity with minimal modification of theras protein. Upon treatment of theras protein with [¹²⁵I] Nal and an Iodobead, radioactivity was initially incorporated into a 22 kDa species with a pl of 5.2, then predominantly into a 23 kDa species with a pl of 5.4. The specific activity of [¹²⁵I]ras was 6×10⁶ cpm/pmol totalras protein. Iondination did not alter the biological activity of theras protein as judged by its ability to bind GTPS and induced maturation ofXenopus laevis oocytes. It is concluded that while iodination alters the apparent molecular weight and pI ofras, presumably by the oxidation of one or more classes of amino acids, this does not affect the biological function of the protein. Theras protein, radioactively-labelled with iodine using the Iodobead method, should be suitable for studies of protein-protein interactions involvingras. Treatment of iodinatedras with the chemical cross-linking agent disuccinimidyl suberate revealed the presence of several minor high molecular weight protein species. This result shows that, in a dilute solution of purifiedras protein, the monomeric form is in equilibrium with small amounts of polymeric forms.Abbreviations DSS Disuccinimidyl Suberate - GTPS Guanosine 5-[-thio] triphosphate - ATPS Adenosine 5[-thio] Triphosphate     

Distinct Responses of Osphradial Neurons to Chemical Stimuli and Neurotransmitters in Lymnaea stagnalis L.

1. In Lymnaea stagnalis L. (Pulmonata, Basommatophora) the neurons in the osphradium were visualized by staining through the inner right parietal nerve by 5,6-carboxyfluorescein (5,6-CF). Three types of neurons were identified: three large ganglionic cells (GC1-3; 80–100 m), the small putative sensory neurons (SC; 20 m) and very small sensory cells (3–5 m).2. The ganglionic and putative sensory neurons were investigated by whole cell patch-clamp method in current-clamp condition. The three giant ganglionic neurons (GC1-3) located closely to the root of osphradial nerve, had a membrane potential (MP) between –30 and –70 mV and showed tonic or bursting activities. The small putative sensory cells (SCs) scattered throughout the osphradial ganglion, possessed a MP between –25 and –55 mV and showed an irregular firing pattern with membrane oscillations. At resting MP the GC1-3 cells were depolarized and increased the frequency of their firing, while the SCs were hyperpolarized and inhibited by NaCl (10^–2 M) and L-aspartate (10^–5 M) applied to the osphradium.3. 5-Hydroxytryptamine (5HT, 10^–6 M), -aminobutyric acid (GABA; 10^–6 M) and the GABA_B agonist baclofen (10^–6 M) depolarized the neurons GC1-3 and increased their firing frequency. In contrast, on the GC1-3 neurons, acetylcholine (Ach; 10^–6 M) and FMRFamide (10^–6 M) caused hyperpolarization and cessation of the firing activity. The 5HT effect was blocked by mianserin (10^–6 M) but picrotoxin (10^–5 M) failed to block the GABA-induced effect on the GC1-3 cells.4. The small putative sensory neurons (SCs) were excited by Ach (10^–6 M) and 5HT (10^–6 M) but were inhibited by GABA (10^–6 M). FMRFamide (10^–6 M) had a biphasic response. The Ach effect was blocked by hexamethonium (10^–6 M) and tetraethylammonium (10^–6 M), indicating the involvement of nicotinic cholinergic receptors.5. The distinct responses of the two populations of osphradial neurons to chemical stimuli and neurotransmitters suggest that they can differently perceive signals from environment and hemolymph.     

Local Perfusion of Nicotine Differentially Modulates Somatodendritic Dopamine Release in the Rat Ventral Tegmental Area After Nicotine Preexposure

We examined the effects of nicotine perfusion into the ventral tegmental area (VTA) on extracellular dopamine (DA) levels in rats using in vivo microdialysis. Local perfusion with nicotine for 80 min (10–100 M) modestly increased (105–131% of basal) the extracellular DA levels in the VTA of rats that had been pretreated with saline for 5 days. In animals that had been pretreated with nicotine for 5 days (0.3 mg/kg, s.c.), perfusion with nicotine for 80 min (10–100 M) dose-dependently increased the extracellular DA levels in the VTA of rats and did so to a greater extent than in saline-pretreated animals (125–171% of basal). Co-perfusion through the dialysis probe with 100 M mecamylamine, a nonselective nicotinic acetylcholine receptor (nAChR) antagonist, or 100 M dihydro--erythroidine, a high affinity and competitive nAChR antagonist, attenuated the enhancement of extracellular DA levels produced by 100 M nicotine alone. These results suggest that local nicotine challenge potentiated the somatodendritic DA release after nicotine preexposure by stimulation of high-affinity nAChRs in the VTA.     

Copper and dyes enhance laccase production in γ-proteobacterium JB

Laccase production in gamma-proteobacterium JB was enhanced 13-fold by adding 0.1 mM CuSO(4) 24 h after the onset of growth. Ethidium bromide (2.5 microM), Malachite Green, Phenol Red and Thymol Blue (10 microM each) enhanced laccase production 17-, 19-, 4- and 2-fold, respectively. Among the fourteen aromatic/organic compounds tried, p-aminobenzoic acid and an industrial effluent, from where the organism was isolated, showed 1.2- and 1.26-fold increases in production.     

Comparative study of inhibitory effects by murine interferon γ and a new bisphosphonate (alendronate) in hypercalcemic,nude mice bearing human tumor (LJC-1-JCK)

The inhibitory effect of murine interferon (muIFN) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), muIFN was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.     

Biochemical and immunocytochemical identification of conglutin γ, a lupin seed lectin,in the roots of young germinating seedlings

Summary Polyclonal antibodies directed against polypeptide epitopes of conglutin , a glycosylated lectin in lupin seed, have been used to identify and quantify this protein in root extracts of germinating lupins. The highest conglutin content was found in protein extracts of root elongation zones after 5 to 7 days germination. Root conglutin showed the same subunit composition, glycosylation pattern, isoelectric point, and lectin activity as the cotyledonary one. Immunolocalization experiments on root thin sections demonstrated that conglutin is chiefly present in the intercellular spaces of the cortical parenchyma, where it forms large aggregates. Labelling of the Golgi complexes and the area between the plasmalemma and cell wall revealed the conglutin pathway from post-synthetic processing to excretion via the secretory system.Abbreviations EDTA ethylene diamino tetraacetic acid - IEF isoelectric focusing - LRW London resin white - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - P_i inorganic phosphate - SDS sodium dodecylsulphate - TEM transmission electron microscopy     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.     

Different pattern of T cell receptor δ gene rearrangement in tumour-infiltrating lymphocytes and peripheral blood in patients with solid tumours

Tumor-infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) from four patients with renal-cell carcinoma (three paired with blood), two colon carcinomas (both paired with blood) and two melanomas (blood was not available) were analysed for the T cell receptor (TCR) gene repertoire. Polymerase chain reaction analysis, employing a panel of specific primers for TCR gene segments, showed different gene rearrangement patterns in TIL and PBL in all patients. Simultaneous analysis of TIL and PBL revealed the presence of lymphoid cells in the tumour tissue that were not present in the periphery. These results demonstrate that, although tumor-infiltrating lymphocytes contain / T cells within the range observed in peripheral blood, these cells differ from those in peripheral blood in their gene repertoire and this may account for selective accumulation or/and in situ amplification of / lymphocytes at the tumour site, indicating a unique type of host reaction against tumors.     

Effect of γ radiation on physiological parameters of the ethanolic fermentation

This work examines the efficacy of radiation in reducing the viability of certain contaminating bacteria of sugar-cane must and the consequential beneficial effect of lethal doses of radiation on some physiological parameters of the yeast-based ethanolic fermentation. The must from sugar-cane juice was inoculated with different bacteria that usually contaminate the must in the production facilities: Bacillus and Lactobacillus. The contaminated must was irradiated at 2.0, 4.0, 6.0, 8.0 and 10.0 kGy of gamma radiation. The population density of the bacteria in the irradiated must was recorded. Ethanolic fermentation by yeast (Saccharomyces cerevisiae) was carried out and the total acidity, the volatile acidity and the organic acids (lactic and acetic) produced during the fermentation were determined. The ethanol yield was also recorded. The treatment with radiation reduced the population of the contaminating microorganisms of the sugar-cane must. The acidity and the organic acids (lactic and acetic) produced during the fermentation decreased as the dose of radiation applied to the must increased. It is concluded that irradiation was efficient in decontaminating the sugar-cane must and improved the biochemical parameters of the ethanolic fermentation, including the ethanol yield by 2%.     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon (IFN) and tumour necrosis factor . A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN, IFN treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Alterations in the expression of the β-cytoplasmic and the γ-smooth muscle actins in hypertrophied urinary bladder smooth muscle

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle -actin, shows over 200% increase in the -actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in -actin, since the 2D gel electrophoresis does not distinguish -smooth muscle actin from -cytoplasmic actin. The bladder smooth muscle -actin and the smooth muscle -actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the -cytoplasmic actin (37%) and a concomitant diminution in the -cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.