Enzymatic synthesis of two fucose-containing glycolipids with fucosyltransferases of human serum.

Lacto-N-neotetraosylceramide incubated with human serum fucosyltransferase preparations gave rise to two fucoglycolipids. The faster migrating fucoglycolipid I on the basis of its thin-layer chromatographic mobility, susceptibility to alpha(1 leads to 2) fucosidase from Trichomonas foetus, radio-immunoprecipitation with Ulex europeus lectin and studies with Oh (Bombay) sera was identified as H-active glycolipid (H-I). The most probable structure of fucoglycolipid II should be that with fucose linked alpha(1 leads to 3) to N-acetylglucosamine. Lactosylceramide, ceramide trihexoside and globoside were not substrates for human serum fucosyltransferases. Lacto-N-neotetraosyl ceramide served as a fucose acceptor for all serum preparations tested while asialoganglioside was a substrate only when serum preparations containing H-gene dependent alpha-2-L-fucosyltransferase were used. With asialoganglioside only one radioactive reaction product was formed.     

Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.     

Transient kinetics of yeast growth in batch and continous culture with an inhibitory carbon and energy source

Growth and acetate metabolism by Candida utilis (ATCC 9226) is reported for both acetate- and zinc-limited cultures in defined media. Acetate concentrations were varied from suboptimal to inhibitory levels in both types of media in differential shake flask culture and in batch and continuous cultures in stirred fermentors. Transient responses of steady-state cultures to small or large additions of concentrated sodium acetate, or to shifts in dilution rate or inlet acetate concentration are compared with one another and with simple mathematical models of growth and acetate metabolism. Exponential growth was observed during unrestricted growth (differential shake flask and batch cultures) with both types of media. Addition of acetate during unrestricted growth always caused lags and for larger pulses, lower specific growth rates were observed after exponential growth resumed. Inhibition by high acetate concentrations was much greater in acetate–limited than in zinc–limited cultures. During restricted growth (steady-state, continuous cultures), high acetate concentrations again consistently caused growth lags but stimulated, inhibited, or temporarily stopped acetage uptake. Qualitative agreement between the predictions of a simple mathematical model of acetate inhibition fitted to differential shake flask data and the observed transient data was surprisingly good.     

Effect of ricin on biosynthesis of myeloma protein (IgA) and general cellular proteins in MOPC-315 cells.

Ricin, a phyto-toxin from the seeds of Ricinus communis, preferentially inhibits synthesis of myeloma protein by MOPC-315 cells as compared to the synthesis of general proteins. In contrast, cycloheximide inhibited equally both myeloma protein synthesis as well as general protein synthesis. These results suggest that proteins synthesized near or on cell membranes are more sensitive to Ricin which binds to the acceptor molecule on the plasma membrane.     

Trans fatty acids induce apoptosis in human endothelial cells.

The present study was designed to investigate the hypothesis that trans fatty acids can induce apoptosis of human umbilical vein endothelial cells (HUVEC). To test this hypothesis apoptosis was measured in HUVEC treated with 0.1, 1.0 or 5.0 mM trans elaidic acid (t-18:1) or linoelaidic acid (t,t-18:2) for 24 hours. For the detection of apoptosis, TdT-mediated dUTP nick end labelling assay (TUNEL), cell binding of annexin V and propidium iodide uptake were measured. Active Caspase-3 and cleaved PARP (poly-ADP-ribose polymerase) were also measured in the cell lysate. Moreover, cellular ability to produce ROS (reactive oxygen species) was measured by DCF fluorescence Both acids studied induce both early (annexin-positive cells) and late stages of apoptosis (cells stained by propidium iodide) in a dose-dependent manner. Also the appearance of TUNEL-positive cells was induced by both trans fatty acids tested, in a dose dependent manner. Both trans acids induce apoptosis through their effect on Caspase-3 activity and on intracellular ROS production. It is worth emphasising that linoelaidic acid proved to be a more potent inducer of apoptosis and ROS production in endothelial cells than elaidic acid. The present studies suggest that trans fatty acids may play a role in damaging and death of vascular endothelial cells in atherosclerosis.     

Introduction of tyramide signal amplification (TSA) to pre-embedding nanogold-silver staining at the electron microscopic level.

The tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis-Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis-side of the Golgi apparatus in both the TSA and the simple protocol.     

New species of Neophyllobius and Tycherobius (Acari, Camerobiidae) from turkey

Two new species of Neophyllobius (Acari, Camerobiidae) viz. N. populus sp. n., N. karabagiensis sp. n., and a new species of Tycherobius (Acari, Camerobiidae), Tycherobius dazkiriensis sp. n., are described from Turkey and illustrated based on females and a protonymph. A key to the known species of Tycherobius is presented.     

A characteristic pharmacological action of ‘Yang-invigorating’ Chinese tonifying herbs: Enhancement of myocardial ATP-generation capacity

In order to investigate the pharmacological basis of ‘Yang-invigorating’ action, the effect of oral treatment with the methanolic extract of ‘Yang-invigorating’ herbs on ATP-generation capacity was examined, using heart homogenates prepared from herb-pretreated mice. Tonifying (i.e., health-promoting) herbs of other functional categories were also included for comparison. The results indicated that ‘Yang-invigorating’ Chinese tonifying herbs could invariably enhance myocardial ATP-generation capacity, with the extent of stimulation varying among the herbs. In contrast, ‘Yin-nourishing’ herbs either did not stimulate or even decreased myocardial ATP-generation capacity. While ‘Qi-invigorating’ herbs produced variable effects on myocardial ATP-generation capacity, most of the ‘blood-enriching’ herbs did not cause any significant changes. The results obtained from studies using myocardial mitochondrial fractions isolated from herb-pretreated mice suggest that ‘Yang-invigorating’ herbs might speed up ATP generation by increasing mitochondrial electron transport. The ensemble of results has provided evidence for the first time to support the pharmacological basis of ‘Yang invigoration’ in Chinese medicine.