Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 μM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of β-oxidation might be correlated with the lower level of acetylcholine synthesis.     

Antifungal effect of CopA3 monomer peptide via membrane-active mechanism and stability to proteolysis of enantiomeric d-CopA3

In our previous study, coprisin, a 43-mer defensin-like peptide, was derived from the dung beetle, Copris tripartitus, and a 9-mer CopA3 (monomer), truncated coprisin analog peptide, was designed. However, the antifungal effects of CopA3 are not known yet. In this study, the antifungal activity and mechanism of CopA3 were investigated and to develop a more effective antimicrobial peptide under physiological conditions, the enantiomeric d-CopA3 was designed. l- and d-CopA3 had a similar antifungal activity without chiral selectivity, and their activity was more potent than that of melittin used as a positive control. Furthermore, l- and d-CopA3 did not even show any hemolysis against human erythrocytes. Membrane studies using propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], suggested that the antifungal effect of l- and d-CopA3 was due to the membrane-active mechanism, by contrast with coprisin possessing apoptotic mechanism without membrane permeabilization. Finally, the proteolytic resistance and antifungal activity of l- and d-CopA3 against trypsin was analyzed by HPLC and colony count assay. The results showed that only d-CopA3 maintained a potent antifungal activity despite the proteolytic condition. Therefore, this study suggests that d-CopA3 has potential as a novel antimicrobial agent.     

δ-Aminolevulinate dehydratase from Raphanus sativus cotyledons

Phytochrome induces δ-aminolevulinate dehydratase (ALAD) activity in radish seedling cotyledons under continuous far red light. Analysis of the enzymatic activity in etioplasts vs total activity shows a constant ALAD level in these organelles (10 %) in etiolated seedlings. In far red irradiated seedlings, the percentage of enzyme detected into etioplasts increases up to 45 % of the total. Comparative kinetic studies of ALAD activity detected in the cytoplasm and the etioplasts indicate an increase in both compartments with a maximum value reached respectively at 96 and 120 hr from sowing. Treatment with cycloheximide shows a very fast abolition of cytoplasmic ALAD activity which is always correlated to an etioplast decrease with a time shift of ca 24 hr. Erythromycin acts only on the cytoplasmic level of ALAD, and only for far red irradiated seedlings, with an increase of activity twice the level detected in untreated ones. This unexpected effect is discussed.     

OCCURRENCE OF PHYIWATED CHLOROPHYLL c IN ISOCHRYSIS GALBANA AND ISOCHRYSIS SP. (CLONE T-ISO) (PRYMNESIOPHYCEAE)1

The pigment composition of two clones of Isochrysis galbana Parke (CCMP 1323 and CCAP 927/1), and Isochrysis sp. (clone T-ISO) was analyzed by high-performance liquid chromatography using a polymeric octadecylsilica column. Fluorescent peaks with retention times higher than chlorophyll a were detected for all three clones. The corresponding pigments were isolated and characterized in terms of their visible absorbance and fluorescence spectra. The pigments were similar to phytol-substituted chlorophyll c, previously isolated from Emiliania huxleyi (Lohm.) Hay and Mohler and other species containing chlmophyll c₃. The presence of phytol-substituted chlorophyll c in I. galbana which lacked chlorophyll c₃, increases the diversity of chlorophyll patterns for the Haptophyta, which can be grouped, at present, into six different pigment types. This is the jrst observation of a haptophyte containing the apolar phytylated chlorophyll c-like pigment but lacking chlorophyll c₃.     

The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Purification of Human Erythrocyte Transglutaminase by Immunoaffinity Chromatography

Human erythrocyte transglutaminase was purified using a reusable immunoaffinity column prepared from a monoclonal antibody described previously (Birckbichler et al., Hybridoma, 4, 179–186, 1985). The purified TGase was catalytically active and exhibited a single band of apparent M_r = 85, 000 on SDS-PAGE and Western blotting. The amino acid composition of the enzyme was determined. The amino terminus was blocked, and the carboxy-terminal residue appeared to be isoleucine.     

Cyclosporine A and PSC833 inhibit ABCA1 function via direct binding

ATP-binding cassette protein A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL). However, the detailed mechanism of HDL formation remains unclear; in order to reveal it, chemicals that specifically block each step of HDL formation would be useful. Cyclosporine A inhibits ABCA1-mediated cholesterol efflux, but it is not clear whether this is mediated via inhibition of calcineurin. We analyzed the effects of cyclosporine A and related compounds on ABCA1 function in BHK/ABCA1 cells. Cyclosporine A, FK506, and pimecrolimus inhibited ABCA1-mediated cholesterol efflux in a concentration-dependent manner, with IC₅₀ of 7.6, 13.6, and 7.0 μM, respectively. An mTOR inhibitor, rapamycin also inhibited ABCA1, with IC₅₀ of 18.8 μM. The primary targets for these drugs were inhibited at much lower concentrations in BHK/ABCA1 cells, suggesting that they were not involved. Binding of [³H] cyclosporine A to purified ABCA1 could be clearly detected. Furthermore, a non-immunosuppressive cyclosporine, PSC833, inhibited ABCA1-mediated cholesterol efflux with IC₅₀ of 1.9 μM, and efficiently competed with [³H] cyclosporine A binding to ABCA1. These results indicate that cyclosporine A and PSC833 inhibit ABCA1 via direct binding, and that the ABCA1 inhibitor PSC833 is an excellent candidate for further investigations of the detailed mechanisms underlying formation of HDL.     

Intestinal caveolin-1 is important for dietary fatty acid absorption

How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound ³H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the ³H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the ³H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with ³H-oleate or ³H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed ³H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1 mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.