Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids,arachidonic acid,and GABA

In rat brain slices the synthesis of [³H]phosphoinositides and the production of [³H]inositol monophosphate (IP₁) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [³H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A₂. Arachidonic acid caused a rapid and dose-dependent inhibition of [³H]phosphoinositide synthesis and of NE-stimulated [³H]IP₁ production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A₂ with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A₂ were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A₂. Further studies were carried out in slices that were prelabelled with [³H]inositol in an attempt to separate modulatory effects on [³H]phosphoinositide synthesis and agonist-stimulated [³H]IP₁ production. Several excitatory amino acid agonists inhibited NE-stimulated [³H]IP₁ production. This inhibitory inter-action could be due to impaired synthesis of [³H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [³H]IP₁ production, indicating that continuous regeneration of [³H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.Abbreviations AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid AP₄ dl-2-amino-4-phosphonobutyric acid - BPB bromphenacyl bromide - BSA bovine serum albumin - CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - DFMO -difluoromethylornithine - DIDS diisothiocyanotostilbene-2,2-disulfonic acid - EGTA ethyleneglycol-bis-N - N, N N-tetraacetic acid - GABA -aminobutyric acid - GDEE glutamate diethyl ether - -GG -glutamylglycine - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - NDGA nordihydroguaiaretic acid - NE norepinephrine - NMDA N-methyl-d-aspartate     

Interaction of alpha-N-Acetyl-beta-endorphin and calmodulin

Acetylation at the -amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide -endorphin, -N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for -N-acetyl--endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like -endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to -endorphin, suggest that residues 14–24 exhibit -helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 µM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the -amino terminal of -endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, -endorphin and the -N-acetylated peptide behave very similarly with respect to calmodulin association.Portions of this work are in partial fulfillment of the requirements for the Ph.D. degree from Vanderbilt University.     

Biosynthesis of Territrems by Aspergillus terreus

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.     

Production and characterization of a monoclonal antibody cross-reactive with most group A trichothecenes

A monoclonal antibody cross-reactive with most group A trichothecenes was produced by fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with 3-acetyl-neosolaniol-hemisuccinate conjugated to bovine serum albumin. One stable clone, H159B1D5, which produced monoclonal antibody that bound with both T-2 toxin and diacetoxyscirpenol (DAS) was obtained after subcloning. Enzyme-linked immunosorbent assay (ELISA) revealed that the antibody belongs to the immunoglobulin G1 (kappa chain) isotype and had binding constants of 2.81 x 10(9), 1.05 x 10(9), and 1.57 x 10(8) liters per mole for T-2 tetraol tetraacetate, T-2 toxin, and DAS, respectively. The relative cross-reactivities of the antibody with T-2 tetraol tetraacetate, T-2 toxin, and DAS were 200, 100, and 20, respectively, with tritiated T-2 toxin as the marker ligand. The relative cross-reactivities for the above toxins were 667, 100, and 73, respectively, with tritiated DAS as the marker ligand. No cross-reaction with HT-2 and deoxynivalenol triacetate was observed in either system. By using this monoclonal antibody, an indirect ELISA for analysis of T-2 toxin was also developed. The linear portion of the standard curve for analysis of T-2 toxin in each analysis by radioimmunoassay and ELISA was in the range of 0.1 to 2 ng and 0.05 to 1.0 ng, respectively.     

Effects of traces of rare earth elements on the protozoanBlepherisma and on mice

The growth of the protozoanBlepherisma is stimulated by Lanthanum (La) at concentrations as low as 0.32 ppm. In mice Yttrium (Y) and Ytterbium (Yb) are absorbed, accumulated, and metabolized. Both rare earth elements (RE) exhibit a high affinity for teeth and bones, accumulation occurs and metabolism is slow. In the livers of RE-exposed mice, concentrations are variable. The liver is apparently capable of absorbing and discharging RE in a manner depending on metabolic activity. The main route of discharge for ingested REs is the alimentary canal. Exposure of pregnant mice to RE leads to rapid placental transfer of RE; 14.1% of the total amount of RE administered was detected in newborn mice. Young, developing organisms appear to be especially susceptible to RE accumulation.     

Biochemical Mapping of Peptidyl-Glycine α-Amidation Activity in the Rat CNS

Peptidyl-glycine alpha-amidation enzyme activity has been measured in 36 nuclei or areas in the rat CNS and pituitary using D-Tyr-Phe-Gly as the substrate. The distribution of this enzyme is highly uneven, with highest activity levels (greater than 30 pmol/mg of protein/h) in hypothalamic nuclei, substantia grisea centralis, and nucleus ruber; moderate activity levels (10-30 pmol/mg of protein/h) in globus pallidus, septum, midbrain, pons, medulla oblongata, and cervical spinal cord; and low activity levels (1-10 pmol/mg of protein/h) in other telencephalic and thalamic structures. Almost no alpha-amidation activity (less than 0.5 pmol/mg of protein/h) was detected in cerebellar cortex. The Km values in several brain regions are of the same order.     

Characterization of lymphoid tumors induced by a recombinant murine retrovirus carrying the avian v-myc oncogene. Identification of novel (B-lymphoid) tumors in the thymus

Lymphoid tumors induced by a recombinant murine retrovirus carrying the v-myc oncogene of avian MC29 virus were characterized. The Moloney murine leukemia virus myc oncogene (M-MuLV (myc], carried by an amphotropic MuLV helper, induced tumors in NIH Swiss and NFS/N mice after a relatively long latency (8 to 24 wk). Tumor masses appeared in the thymus, spleen, and lymph nodes. Flow cytometry of the tumor cells indicated that approximately 50% were positive for Thy 1.2. Most of these tumors also expressed one or more other cell surface markers of thymocytes and mature T cells (CD4, CD8). Southern blot hybridization revealed genomic rearrangements for the TCR beta genes. The TCR beta analysis suggested that the M-MuLV(myc)-induced Thy 1.2+ tumors were derived from somewhat less mature cells than tumors induced by M-MuLV, which is a classical non-acute retrovirus lacking an oncogene. The remainder of the M-MuLV(myc)-induced tumors were Thy 1.2-, but they were positive for Ly-5 (B220) and also for MAC-2. The Thy 1.2- tumors were characteristically located in the thymus. However, they were negative for TCR beta gene rearrangements. Some, but not all, of the Thy 1.2- tumors contained rearrangements for Ig genes. Additionally, they typically expressed mRNA specific for B but not for T cells. Thus, these thymic tumors had characteristics of the B cell lineage. Tumor transplantation experiments demonstrated that the Thy 1.2- tumor cells could reestablish in the thymus and spleen of irradiated hosts, and low level expression of the Thy 1 molecule was observed in the thymus but not the spleen on the first passage. After serial passage, one Thy 1- tumor altered its cell surface phenotype to Thy 1low B220-.     

Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1

The stability (stb) locus of IncFII plasmid NR1 was mapped to a 1700 base-pair NaeI-TaqI restriction fragment. A series of unstable plasmids that contained insertion, deletion, and point mutations that inactivated the stability function was isolated. The unstable point mutants examined were all stabilized (complemented) in trans by a copy of the wild-type stb locus, suggesting that the mutations had inactivated diffusible gene products. The nucleotide sequence of the stb locus contained two tandem open reading frames, designated stbA and stbB, that encoded essential trans-acting protein products with predicted sizes of 36,000 Mr and 13,000 Mr, respectively. A third open reading frame, stbC, that could encode a peptide of 8000 Mr was contained within stbB in the complementary DNA strand. Plasmid-encoded proteins of 36,000 Mr and 13,000 Mr were identified in minicell experiments as the products of stbA and stbB, respectively. Unstable deletion mutants that retained the promoter proximal region of the stb locus upstream from stbA but had deleted both stbA and stbB were stabilized in trans by plasmids that could supply StbA and StbB. In contrast, deletion mutants that had lost the stbAB promoter region were not complemented in trans, indicating that this region contained an essential cis-acting site (or sites). Unlike some other loci that mediate stable plasmid inheritance, cloned copies of the wild-type stb locus of NR1 did not exert strong incompatibility (i.e. trans destabilization) against other stb+ derivatives of plasmid NR1 present in the same cell.     

钙调素抑制剂——三氟拉嗪对肿瘤细胞增殖和微管组装的影响

本文用钙调素抑制剂——三氟拉嗪处理人胃癌MGC-803细胞,用免疫荧光细胞化学方法,放射免疫法和速流荧光分析等方法研究了钙调素对细胞增殖,环核苷酸代谢及微管组装,有丝分裂等细胞功能的调节作用。实验结果表明,TFP明显地抑制了人胃癌细胞的增殖,这种抑制增殖的作用,具有剂量和时间依赖关系,细胞群体中G_1期细胞增多,S期细胞下降,DNA合成明显地受到抑制。TFP处理的胃癌细胞仅在短时间内(5'-30')cAMP含量升高,cGMP浓度降低,cAMP/ ??cGMP比值比对照组高4.4倍,但此后环核苷酸含量又很快恢复到对照组水平。本实验还观察到TFP处理后的MGC-803细胞胞质铺展,细胞形态的改变与胞质微管的分布有密切联系,实验结果表明TFP加强了人胃癌细胞MTOC对微管的组装能力,使微管分布得到恢复,微管纤维呈放射状延伸到细胞边缘,充满胞浆,使细胞呈现出展平的多边形,趋向于正常上皮细胞形态的变化,本实验结果表明TFP抑制癌细胞增殖及使微管组装加强可能是通过对CaM活性的抑制作用。此结果有助于说明转化细胞内钙调素的变化,可能是与转化细胞增殖失控和胞质微管消退有关。     

An electronic mechanism in the action of drugs, ATP, transmitters and other cardinal adsorbents. II. Effect of ouabain on the relative affinities for Li+, Na+, K+, and Rb+ of surface anionic sites that mediate the entry of Cs+ into frog ovarian eggs

Ouabain enhanced the inhibitory effects of Li+, Na+, and K+ on the rate of Cs+ permeation into frog ovarian eggs while it reduced the inhibiting effect of Rb+. The data agree with earlier demonstrated effects of ouabain on the rank order of selective accumulation of the five alkali-metals in frog muscles and on the relative effectiveness of glycine, Li+, Na+, K+, Rb+, and Cs+ in inhibiting the rate of entry of Cs+ into frog sartorius muscle. In all three cases, the ouabain behaved as an electron-donating cardinal adsorbent (EDC) causing a rise of the electron density (c-value) of the beta- and gamma-carboxyl groups in the cell cytoplasm (for selective accumulation) and on the cell surface (for selective ion permeation). Explanations based on the association-induction hypothesis were offered why an EDC like ouabain does not initiate cell activation (like veratridine does) and why Ca++ and tetradotoxin delays or inhibits physiological and artificial cell activation.