γ-Glutamyl conjugation of 5-hydroxytryptamine (serotonin) in the earthworm (Lumbricus terrestris)

The conversion of 5-hydroxytryptamine to several potential metabolites was examined in the annelid earthworm (Lumbricus terrestris). 5-hydroxytryptamine and some related amines were found to be present in several tissues of the earthworm. Injection of 5-hydroxytryptamine into the body cavity of the earthworm resulted in the production of a -glutamyl conjugate of 5-hydroxytryptamine. Incubations of the anterior nerve cord of the earthworm resulted in the accumlation of considerable amounts of 5-hydroxytryptamine and -glutamyl 5-hydroxytryptamine in the incubation medium. The earthworm did not produce any N-acetyl 5-hydroxytryptamine and only very little 5-hydroxyindoleacetic acid. Experiments involving the injection of radiolabeled 5-hydroxytryptamine or coninjection of radiolabeled glutamic acid with unlabeled 5-hydroxytryptamine into the earthworm resulted in the production of radiolabeled -glutamyl 5-hydroxytryptamine. This work demonstrates that the enzymatic conversion of 5-HT in the earthworm is markedly different from that of vertebrates and insects.     

The evolution of pathways for aromatic hydrocarbon oxidation inPseudomonas

The organisation and nucleotide sequences coding for the catabolism of benzene, toluene (and xylenes), naphthalene and biphenylvia catechol and the extradiol (meta) cleavage pathway inPseudomonas are reviewed and the various factors which may have played a part in their evolution are considered. The data suggests that the complete pathways have evolved in a modular way probably from at least three elements. The commonmeta pathway operons, downstream from the ferredoxin-like protein adjacent to the gene for catechol 2,3-dioxygenase, are highly homologous and clearly share a common ancestry. This common module may have become fused to a gene or genes the product(s) of which could convert a stable chemical (benzoate, salicylate, toluene, benzene, phenol) to catechol, thus forming the lower pathway operons found in modern strains. The upper pathway operons might then have been acquired as a third module at a later stage thus increasing the catabolic versatility of the host strains.     

Identification of N-terminal helix capping boxes by means of 13C chemical shifts

Summary We have examined the ¹³C and ¹³C chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with and clustering around 94±15° and 167±5°, respectively. This is manifested by a 1–2 ppm upfield shift of the ¹³C resonance and a 1–4 ppm downfield shift of the ¹³C resonance, relative to their random coil values, and is mainly associated with the unusually large value of . The residues following the N-cap residue exhibit downfield shifts of 1–3 ppm for the ¹³C resonances and small upfield shifts for the ¹³C ones, typical of an -helix.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

Cytosolic 5′-nucleotidase/nucleoside phosphotransferase: A nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5′-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2′,3′-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5′-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD₅₀ = 0.32 μM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5′-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5′-nucleotidase is not essential for the activation of this nucleoside analog.     

Regional Distribution and Quantitative Measurement of the Phosphoinositidase C-Linked Guanine Nucleotide Binding Proteins G1α and Gqα in Rat Brain

Abstract: Levels of the guanine nucleotide binding proteins G₁₁α and G_qα, which produce receptor regulation of phosphoinositidase C., were measured immunologically in 13 regions of rat central nervous system. This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis conditions that can resolve G_qα and G₁₁α. In all regions examined, G_qα was more highly expressed than G₁₁α. Ratios of levels of G_qα to G₁₁α varied between the regions from 5:1 to 2:1. Quantitative measurements of the levels of G_qα and G₁₁α in each region were obtained by comparison with known amounts of purified liver G_qα and G₁₁α and with E. coli expressed recombinant G_qα. Areas that expressed G_qα highly included olfactory bulb (930 ng/ mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein). More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein). Gna was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of G_qα. A third polypeptide could be identified by the CQ antisera in all brain regions. The possibility that this polypeptide is the α subunit of G₁₄ is discussed.     

Effect of Inhibitors of Eicosanoid Metabolism on Release of [3H]Noradrenaline from the Human Neuroblastoma, SH-SY5Y

Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [³H]noradrenaline ([³H]NA) from the human neuroblastoma clone SH-SY5Y with IC₅₀ values of 10, 15, and 30 μ M , respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 μ M ). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophoreevoked release of [³H]NA.     

Prolactin-Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C-Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL-induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL-induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine, or polymyxin B blocked the PRL-induced increase in cell number with IC₅₀ values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α-phorbol 12,13-didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U-shaped dose-response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose-response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL-induced increase in cell number. Finally, TPA treatment decreased cell-surface binding of ¹²⁵I-PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.     

Diffuse serotoninergic neurohemal systems associated with cerebral and suboesophageal nerves in the head of the Colorado potato beetle Leptinotarsa decemlineata

We analyzed the anatomy of two diffuse neurohemal systems for serotonin in the head of the Colorado potato beetle Leptinotarsa decemlineata by means of immunohistochemistry. One system is formed by axons from two bilateral pairs of neurons in the frontal margin of the suboesophageal ganglion that enter the ipsilateral mandibular nerve, emerge from this nerve at some distance from the suboesophageal ganglion, and cover all branches of the mandibular nerve with a dense plexus of immunoreactive axon swellings. The other system is formed by axons from two large neurons in the frontal ganglion that enter the ipsilateral frontal connectives, emerge from these connectives, and form a network of axon swellings on the labroforntal, pharyngeal, and antennal nerves and on the surface of the frontal ganglion. Immunohistochemical electron microscopy demonstrated that the axon swellings are located outside the neural sheaths of the nerves and hence in close contact with the hemolymph. We therefore suggest that these plexuses represent extensive neurohemal systems for serotonin. Most immunoreactive terminals are in direct contact with the hemolymph, and other terminals are closely associated with the muscles of the mandibles, labrum, and anterior pharynx, as well as with the salivary glands, indicating that these organs are under serotoninergic control.