The Effect of the B Subunit of Cholera Toxin on the Action of Nerve Growth Factor on PC12 Cells

Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [³H]-thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca²⁺ in the medium. ¹²⁵I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.     

Antimicrobial activity of lipoprotein particles containing apolipoprotein Al

Human plasmain vitro inhibits the growth of coagulase negative staphylococci,S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity againstS. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated withS. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation.To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition.These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.     

Effects of physical exercise on the elasticity and elastic components of the rat aorta

To evaluate the effects of exercise on aortic wall elasticity and elastic components, young male rats underwent various exercise regimes for 16 weeks. In the exercised rats, the aortic incremental elastic modulus decreased significantly when under physiological strain. The aortic content of elastin increased significantly and the calcium content of elastin decreased significantly in the exercised group. The accumulated data from the exercised and sedentary groups revealed that the elastin calcium content was related positively to the incremental elastic modulus. We concluded that physical exercise from an early age decreases the calcium deposit in aortic wall elastin and that this effect probably produced in the exercised rats a distensible aorta.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

EPR Spin Trapping Study of the Decomposition of Azo Compounds in Aqueous Solutions by Ultrasound: Potential for Use as Sonodynamic Sensitizers for Cell Killing

Sonodynamic therapy, a promising new approach to cancer treatment, is based on synergistic cell killing by combination of certain drugs (sonosensitizers) and ultrasound. Although the mechanism of sonodynamic action is not understood, the role of free radicals produced from sonosensitizers by ultrasound is implicated. In this work, we studied formation of free radicals during the decomposition of several water-soluble azo compounds by 50 kHz ultrasound in aqueous solutions. Using the spin trap 3, 5-dibromo-4-nitrosobenzene sulfonate (DBNBS) tertiary carbon-centered radicals from 2, 2'-azobis (N,N'-dimethyl-eneisobutyramidine) dihydrochloride (VA-044), 2-(carbamoylazo)-isobutyronitrile (V-30), and 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and CH3 radicals from 1, 1'-azobis (N,N'-dimethylformamide) (ADMF) were detected in argonsaturated solutions and the corresponding oxygen-centered radicals (alkoxyl and peroxyl) from VA-044, V-30, and AAPH were identified using the spin trap 5, 5'-dimethyl-l-pyrroline-N-oxide (DMPO) in aerated sonicated solutions. No free radicals from 4, 4'-dihydroxyazobenzene-3, 3'-dicarboxylic acid, disodium salt (DHAB) could be found in either system. While VA-044 and AAPH could also be readily decomposed by heat (42.5°C and 80°C), V-30 decomposition only occurred in the ultrasound-exposed solutions. The most likely mechanism of decomposition of azo compounds by ultrasound is their thermolysis in the heated shell of the liquid surrounding ca vita ting bubbles driven by ultrasound and/or by pyrolysis inside these bubbles. Experiments using scavengers of ·OH and ·H, which are produced by sonolysis in aqueous solutions, demonstrated that these radicals are not involved in the ultrasound-mediated radical production from the azo compounds. Due to the known cytotoxic potential of free radicals produced from azo compounds, the use of these compounds as ultrasound sensitizers appears to be a promising approach for sonodynamic cell killing.     

Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress

Glycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of certain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild-type plants failed to germinate. At 100 mM NaCl, transformed plants grew well whereas wild-type plants did not do so. The transformed plants tolerated 200 mM NaCl, which was lethal to wild-type plants. After plants had been incubated with 400 mM NaCl for two days, the photosystem II activity of wild-type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild-type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.     

Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B

Abstract: Activation of the N -methyl- d -aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.     

Novel mutation at the initiation codon in the Norrie disease gene in two Japanese families

We have identified a new mutation of Norrie disease (ND) gene in two Japanese males from unrelated families; they showed typical ocular features of ND but no mental retardation or hearing impairment. A mutation was found in both patients at the initation codon of exon 2 of the ND gene (ATG to GTG), with otherwise normal nucleotide sequences. Their mothers had the normal and mutant types of the gene, which was expected for heterozygotes of the disease. The mutation of the initiation codon would cause the failure of ND gene expression or a defect in translation thereby truncating the amino terminus of ND protein. In view of the rarity and marked heterogeneity of mutations in the ND gene, the present apparently unrelated Japanese families who have lived in the same area for over two centuries presumably share the origin of the mutation.     

Optimal strategy of plant antiherbivore defense: Implications for apparency and resource-availability theories

Plants produce chemicals as methods against animal herbivory. Such chemical defenses are classified into two major categories: (i) quantitative defenses with massive production of indigestible substances; and (ii) qualitative defenses with production of poisonous substances. A mathematical model was developed that identified factors that favored the evolution of quantitative defenses. Selecting an annual plant for simplicity, the allocation of photosynthetic production between growth substances and defense substances was considered. If the plant invests more in defense substances, it can protect itself more efficiently from herbivory but with a reduced growth rate. If it invests more in growth substances, the contrary holds. Using Pontoryagin's maximum principle, the following results were obtained: (i) the plant should conduct quantitative defenses when the growth rate (G), reflecting resource-availability, is low and the growth period (T) is long as well; (ii) if the plant invests in quantitative defenses, the optimal proportion of defense substances (χ^*) should be higher asG is smaller, but it is independent ofT; and (iii) the value of χ^* is not monotone for the effectiveness of defense substance (A), but has a maximum at an intermediate value ofA. Predictions of the model partly supported both Feeny's apparency theory, claiming that apparent plants or their parts for herbivores should quantitatively defend themselves, and Coley's resource-availability theory, claiming that plants with rich resources should invest in growth rather than defense.