Photosynthetic Adaptation to High Temperature Associated with Thylakoid Membranes of Synechococcus PCC7002

Photosynthetic adaptation to high temperature was investigatedin intact cells and isolated thylakoid membranes of the cyanobacterium,Synechococcus PCC7002. In intact cells, the thermal stabilityof photosynthesis and photosystem 2-mediated electron transportfrom H₂O to 1,4-benzoquinone changed in concert with growthtemperature. The photosystem 2-mediated electron transport fromH₂O to phenyl-1,4-benzoquinone showed greater thermal stabilityin thylakoid membranes isolated from cells which had adaptedto high temperature than in those from non-adapted cells. Enhancedthermal stability was also observed in the thylakoid membranesin the transport of electrons from H₂O to 2,6-dichlorophenolindophenolbut not in the transport of electrons from diphenylcarbazideto 2,6-dichlorophenolindophenol. These observations suggestthat oxygen-evolving sites acquire enhanced thermal stability,and that factors which are responsible for thermal stabilityremain in isolated thylakoid membranes. (Received October 30, 1992; Accepted December 18, 1992)     

Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli for glutamate 1-semialdehyde (GSA) aminomutase, which is involved in the C5 pathway for synthesis of -aminolevulinic acid (ALA), was cloned onto a multicopy plasmid, pUC18, by the complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of deletion fragments from the initial 3.5-kb chromosomal fragment allowed the isolation of a 1.7-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.7-kb DNA fragment revealed an open reading frame (ORF) that is located downstream from a potential promoter sequence and a ribosome-binding site. The ORF encodes a polypeptide of 429 amino acid residues, and the deduced molecular mass of this polypeptide is 45,043 Da. The amino acid sequence shows a high degree of homology to the HemL proteins from other organisms, and a putative binding site for pyridoxal 5-phosphate is conserved. Correspondence to: Y. Murooka     

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.     

Gene introduction into mouse blastocysts via “pricking”

It is a well-known phenomenon that cultured mammalian cells that have been pricked in the presence of foreign DNA can be transformed. This micromanipulation ‘pricking’ technique was applied to mouse blastocysts to determine whether uptake of exogenous DNA would occur in the embryos. The middle region of the inner cell mass (ICM) was pricked three times in each blastocyst in a medium containing a linearized plasmid DNA. When the 60 treated blastocysts were transferred to the uterine horns of pseudopregnant females, 30 developing fetuses (50%) at the mid-gestation stage were obtained. Twenty-two of the 30 fetuses (73%) had less than 1 copy of the foreign DNA per diploid cell, as revealed by polymerase chain reaction (PCR)-Southern analysis, a sensitive technique combined with Southern blot processing of the PCR products. The 8 other fetuses were negative for the foreign DNA. When blastocysts were pricked in the presence of vector DNA coupling E. coli β-galactosidase (β-gal) gene to a mouse metallothionein-I (MT-I) promoter and assessed for β-gal activity histochemically after 1 and 5 days of culture in the presence of 1 μM CdCI₂, at least 65% of the embryos exhibited β-gal activity mainly in the ICM region. These results indicate that mouse blastocysts can be transfected with a relatively high efficiency after pricking, and that the introduced gene expression occurs. This approach provides a means of mapping the regulatory elements of genes that are active in the mouse blastocyst ICM, and may be useful in investigating the fate of the ICM cells in an intact blastocyst by labeling them via pricking technique. © 1993 Wiley-Liss, Inc.     

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.     

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.