A new method for the separation of the catecholamines by gas-liquid chromatography.
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Ti plasmid mutants derived from Agrobacterium tumefaciens strain Ach5 that induce tumors of abnormal morphology have been analyzed. On tobacco, A. tumefaciens mutant strain LBA4060 induces tumors that specifically give rise to shoots. Shoots continue to grow from in vitro cultured bacteria-free tumor tissue derived from such tumors. The mutant character is shown to be correlated with the insertion of an A. tumefaciens IS element, IS60, into the left arm of the T-region of the octopine Ti plasmid. Evidence is presented showing that IS60 is transferred into the plant cell DNA as part of the T-DNA. A second Ti plasmid insertion mutant A. tumefaciens strain LBA4210, with a Tn904 transposon in the center of the T-region, induces tumors that specifically exhibit a root development on tobacco plants. T-DNA has been detected in sterile amorphous crown-gall tissue derived from these tumors. The transposon Tn904 insertion was shown to result a changed "core" T-DNA. Abnormal tumor morphologies induced by these mutant strains have been observed also on Kalanchoë stems. On tomato plants the mutants induce small unorganized tumors while on Nicotiana rustica unorganized tumors, nearly equal in size to those caused by the wild-type strain have been induced. LBA4060 was shown to be avirulent on Kalanchoë leaves and LBA4210 was weakly virulent. Infection of Kalanchoë leaves or tomato plants with a mixture of separately grown cultures of both mutants resulted in the formation of more or less normal tumors. The exposure of a tomato plant to naphthalene acetic acid (NAA), a synthetic auxin, during development of tumors induced by LBA4060 stimulated tumor formation. Tumor growth induced by LBA4210 was found to be stimulated by kinetin.
Three identifiable giant neurons, which were morphologically and pharmacologically identical, named TAN (tonically autoactive neuron), TAN-2 and TAN-3, were found in line on the dorsal surface of the right parietal ganglion in the suboesophageal ganglia of an African giant snail (Achatina fulica Férussac). The diameters of these neurons were 150-200 microns. They showed regular spontaneous spike discharges at the rate of 30-40 per min. However, the spike discharges of the three neurons were not synchronized. For morphological studies of these neurons, Lucifer Yellow was injected into their somata by pressure. The axonal branches of the three neurons examined extended commonly into the following seven peripheral nerves: left anterior pallial, left posterior pallial, intestinal, anal, right posterior pallial, right anterior pallial and right anterior pallial accessory nerves. The sensitivities of the three neurons examined to the main neurotransmitter candidates and their derivatives were almost identical. These neurons were excited by 5-hydroxytryptamine [the minimum effective concentration (MEC): 10(-5)-10(-4) M] and histamine (MEC: 1-3 X 10(-4) M), and inhibited by dopamine (MEC: 1-3 X 10(-4) M), L-norepinephrine (MEC: 3 X 10(-4)-10(-3) M), L-epinephrine (MEC: 3 X 10(-4) M), GABA (MEC: 10(-5)-10(-4)), acetylcholine (MEC: 1-3 X 10(-4) M) and its derivatives.
1. Cholinergic synapses in the central nervous system of insects contain inhibitory muscarinic receptors whose stimulation by agonists leads to a diminished output of acetylcholine; antagonists, like atropine, facilitate acetylcholine release. 2. The receptors involved appear to be of the M2-subtype. Upon activation of presynaptic receptors a significant reduction of the intrasynaptosomal cyclic AMP level as well as a significantly increased membrane potential was observed. 3. The observed membrane hyperpolarization is apparently not a consequence of a lower cyclic AMP level, thus both effects may offer alternative or synergistical mechanisms for modulating transmitter release.
Conversion of neurotransmitter dopamine into norepinephrine is catalyzed by dopamine beta-hydroxylase (DbH). The reaction requires the presence of both molecular oxygen and a reducing cosubstrate, the assumed physiological cosubstrate being ascorbic acid. We have investigated the ability of a new family of molecules, N-aryl-N'-hydroxyguanidines, to serve as cosubstrates for DbH. N-(4-Methoxyphenyl)-N'-hydroxyguanidine proved to be an efficient reducing agent for DbH. The complete N-hydroxyguanidine moiety was required for activity, as any modification of this function resulted in non-cosubstrate compounds. Moreover, analysis of the products formed from N-(4-methoxyphenyl)-N'-hydroxyguanidine showed that the main oxidation product was a nitrosoimine. Modification of the aromatic para-substituent evidenced an influence of its electronic properties on the catalytic activity whereas steric factors seemed less important. In addition, changing the methoxy-substituent from the para- to the ortho-position led to an inactive compound. Our results demonstrate that N-aryl-N'-hydroxyguanidines are new efficient reducing cosubstrates for DbH and prove that specific interactions with the reducing cosubstrate do take place at the active site of the enzyme.
Cyclic adenosine-monophosphate (cAMP) is one of the major second messenger molecules transmitting extracellular stimuli into short- and long-term changes of intracellular homeostasis. Measurements of cellular cAMP levels are often used to quantify and characterize signaling by G protein-coupled receptors. Current assays for cAMP determination are usually end-point assays involving cell lysis. We have developed a technology to monitor real-time changes of cAMP levels in living cells. This method uses a modified cyclic nucleotide-gated (CNG) Ca(2+) channel which is opened by intracellular cAMP. Thus, changes in cAMP levels are translated into changes in free Ca(2+) which can easily be measured using fluorimetric imaging technologies compatible with high-throughput screening formats. The new assay method was used to characterize the pharmacology of various endogenously and heterologously expressed G protein-coupled receptors and allows for the simultaneous study of G(s), G(i) and G(q)-linked receptors in the same cell population.
Biventricular dilation and severe cardiac dysfunction are observed during septic shock. However, when endotoxemia and vasoconstrictor-masked hypovolemia work in concert in the pathogenesis of shock, the clinical scenario is more adverse compared to one of the insults acting alone. Matrix metalloproteinases (MMPs) are involved in chronic and acute heart failure by degrading the mechanical scaffold of the heart and several intracellular proteins. Therefore, the roles of MMP-2, MMP-9, MT1-MMP, focal adhesion kinase (FAK), and Paxillin in hearts of early multiple organ failure induced by norfenefrine-masked hypovolemia and endotoxemia were investigated in an ovine model. Experimental groups included (1) norfenefrine-masked hypovolemia plus endotoxemia (NMH+ENDO) (n=6), (2) norfenefrine-masked hypovolemia without endotoxemia (NMH) (n=6), (3) recurrent endotoxemia during normovolemia (ENDO) (n=6), and (4) healthy untreated controls (CON) (n=3). Apoptosis was determined by TUNEL-staining. Gel zymography revealed significantly increased MMP-2 activity in NMH+ENDO compared to ENDO and controls. MMP-9 activity was significantly elevated in all experimental groups. MMP-2 was significantly increased at the protein level, while MMP-9 was unaltered. MT1-MMP was not significantly changed in any group. Increased MMP activities were associated with cardiac deterioration. MMP-2/-9 activity and phosphorylated Paxillin (p-Paxillin) expression correlated positively with cardiomyocyte apoptosis. This study underscores the pivotal roles of MMP in acute cardiac dysfunction during early multiple organ failure in combined vasoconstrictor-masked hypovolemic and endotoxemia shock.
Progress in the field of insect neuroendocrinology has been rapid despite the relatively small number of investigators working on insect systems. This progress, in part, reflects the ease of studying insect behavior in the laboratory, and a historical perspective reveals that insect neuroendocrinology has been dominated since its inception by laboratory studies. Recent advances in methodology and a renewed interest in the concept of behavioral state in insects suggest that it might be useful for insect neuroendocrinologists to spend a little more time in the field.
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The expression of a cloned Drosophila octopamine/tyramine receptor (OctyR99AB) is described in Xenopus oocytes. Agonist stimulation of OctyR99AB receptors increased intracellular Ca2+ levels monitored as changes in the endogenous inward Ca2+-dependent chloride current. The receptor is preferentially sensitive to biogenic amines with a single hydroxyl on the aromatic ring. The G-protein, Galphai, appears to be involved in the coupling of the receptor to the production of intracellular calcium signals, since the effect is pertussis-toxin sensitive and is blocked or substantially reduced in antisense knockout experiments using oligonucleotides directed against Galphai but not by those directed against Galphao, Galphaq and Galpha11. The increase in intracellular calcium levels induced by activation of the OctyR99AB receptor can potentiate the ability of activation of a co-expressed beta2-adrenergic receptor to increase oocyte cyclic AMP levels. A comparison of the pharmacological coupling of OctyR99AB to different second messenger systems when expressed in Xenopus oocytes with previous studies on the expression of the receptor in a Chinese hamster ovary cell line suggests that the property of agonist-specific coupling of the receptor to different second messenger systems may be cell-specific, depending upon the G-protein environment of any particular cell type.
Phenol sulfotransferase (PST, EC 2.8.2.1) and glutathione-S-transferase (GST, EC 2.5.1.18), the phase II biotransformation enzymes inactivate many exo- and endogenous compounds. The effect of PST substrates (catecholamines, simple phenols, selected phenolic drugs) and PST products (phenolic sulfates) on GST activity was investigated to identify possible interactions between sulfation and glutathione conjugation in the brain. Two soluble forms of PST and two forms of GST were isolated from monkey (Rhesus macacus) brain cortex. Catecholamines, hypertensive and hypotensive drugs which are sulfated by monkey brain PSTs slightly inhibit the activity of brain GSTs. The greatest inhibitory effect was observed with neurotoxic compounds such as 6-OHDA and manganese. The commonly used analgesic drugs inhibit both GST forms. These enzymes are also inhibited by phenacetin, the precursor of paracetamol, and prototype salicylates such as sodium salicylate and acetylsalicylic acid. The effect of simple phenols and their sulfated metabolites on GST activity varies. The obtained results point to a possible interaction between sulfation and glutathione conjugation in vivo since many physiologically, therapeutically and toxicologically active compounds which are sulfated by brain phenol sulfotransferases may be bound by brain glutathione-S-transferases. These compounds may lose their activity (on being bound to GST) and expose the brain to the toxic electrophiles (by decreasing GST activity).
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