Effects of Prostaglandin E2 and Capsaicin on Behavior and Cerebrospinal Fluid Amino Acid Concentrations of Unanesthetized Rats: A Microdialysis Study

Abstract: Prostaglandin E₂ (PGE₂) delivered to the spinal cord produces an increased sensitivity to noxious (hyperalgesia) and innocuous (allodynia) stimuli. The mechanisms that underlie this effect remain unknown, but a PGE₂-evoked enhancement of spinal neurotransmitter release may be involved. To address this hypothesis, we examined the effect of PGE₂ on CSF concentrations of amino acids and also the modulatory effect of PGE₂ on capsaicin-evoked changes of spinal amino acid concentrations using a microdialysis probe placed in the lumbar subarachnoid space. Amino acids were quantified using HPLC with fluorescence detection. Addition of 1 mM, but not 10 or 100 µM, PGE₂ to the perfusate for a 10-min period (flow rate, 5 µl/min) evoked an immediate increase (80–100%) in glutamate (Glu), aspartate (Asp), taurine (Tau), glycine (Gly), and γ-aminobutyric acid (GABA) concentrations. Similarly, capsaicin infusion (0.1–10 µM) induced a dose-dependent increase in Glu, Asp, Tau, Gly, GABA, and ethanolamine levels. Significant increases in amino acid levels evoked by PGE₂ or capsaicin were associated with a touch-evoked allodynia. The combination of PGE₂ (10 µM) and capsaicin (0.1 or 1.0 µM) at concentrations that individually had no effect together evoked a significant increase (60–100%) in Glu, Asp, Tau, Gly, and GABA concentrations and produced tactile allodynia. These data demonstrate that spinally delivered PGE₂ or capsaicin substantially elevates CSF concentrations of both excitatory and inhibitory amino acids. The capacity of PGE₂ to enhance and prolong capsaicin-evoked amino acid concentrations may be one of the mechanisms by which spinal PGE₂ produces hyperalgesia and allodynia.     

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.     

Solubilization and hydrophobicity test by Triton X-114-partitioning of NADPH-protochlorophyllide oxidoreductase from the unicellular alga Scenedesmus obliquus, mutant C-2A'

NADPH-protochlorophyllide oxidoreductase (PChilde reductase, EC 1.3.1.33), a key enzyme in light-dependent greening and the conversion of etioplasts into chloroplasts was investigated in the the greening mutant C-2A' of the unicellular green alga Scenedesmus obliquus. In the absence of detergent, the solubilization of the enzyme increased with high glycerol concentrations in the buffer. Solubilization capacities of 4 non-ionic or zwitterionic detergents, Triton X-100, CHAPS, octylglucoside and decyl-maltopyranoside, were compared. Due to the addition of these detergents, the enzyme activity in the soluble fraction was increased severalfold. Hydrophobicity of the enzyme was analyzed by Triton X-114 phase partitioning. The protein had a preference for the aqueous phase, but its distribution was strongly influenced by the glycerol concentration of the buffer. These results indicate that the PChlide reductase of the green alga Scenedesmus obliquus is a hydrophobic, membrane-associated enzyme, but not an integral membrane protein.     

Tryptophan Decarboxylase,Tryptamine, and Reproduction of the Whitefly

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine. When the TDC gene was expressed in transgenic tobacco, the 55-kD TDC enzyme and tryptamine accumulated. Bemisia tabaci (sweetpotato whitefly) reproduction on transgenic plants decreased up to 97% relative to controls. Production of tryptamine, its derivatives, or other products resulting from TDC activity may discourage whitefly reproduction and provide a single-gene-based plant protection strategy.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mol PPFD m^-2 s^-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mol PPFD m^-2 s^-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.Abbreviations IPT isopentenyl, transferase - rbcS-3A gene encoding a small subunit protein (SSU) of Rubisco from Pisum sativum - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Analysis of randomly isolated cDNAs from developing endosperm of rice (Oryza sativa L.): evaluation of expressed sequence tags,and expression levels of mRNAs

Using a cDNA library prepared from poly(A)⁺ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, -1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycinerich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.