Responses of Isolated Plant Mitochondria to Photosynthesis Inhibiting Acylanilides

During experiments to elucidate the mode of action of photosynthesis inhibiting acylanilide type herbicides, the effects of various acylanilides on respiration and oxidative phosphorylation of isolated plant mitochondria were studied. The results showed that some acylanilides acted as uncouplers of oxidative phosphorylation: 1) Some stimulated the ADP-limited state 4 respiration of isolated mitochondria depriving them of their respiratory control ability during succinate oxidation. 2) Those which stimulated state 4 respiration interfered with oxidative phosphorylation to degenerate the P/O ratio.

The following relationships between chemical structure of acylanilides and their biological activities were demonstrated: 3) Among various ring-chlorinated propionanilides, the activity of 3′,4′-DCPA was especially prominent. 4) Almost all the side chain-substituted 3′,4′-dichloroacylanilides tested were effective. 5) Both chlorination of the 3 and 4 positions of the aniline moiety and acylanilide bonding were simultaneously required for an acylanilide to produce uncoupling activity. 6) DCMU was less effective than was 3′,4′-DCPA, both in stimulating state 4 and in degenerating the P/O ratio.     

Effect of alcohol drinking and cigarette smoking on neutrophil functions in adults

In recent years, the effects of smoking and excessive alcohol consumption on immune function have been studied, due to a high prevalence of infection or cancer in heavy drinkers, and the combination of smoking and drinking was considered to be a carcinogenic risk. However, the effect of smoking and drinking on systemic immune function has yet to be clearly understood. In this study, we investigated neutrophil functions (reactive oxygen species (ROS) productive activity, phagocytic ability and serum opsonic activity) and their relationship with alcohol consumption or amount of smoking. In total there were 731 male and female adult subjects who participated in the Iwaki Health Promotion Project in 2005. Multiple regression analysis showed a trend of increased ROS production in male subjects and a statistically significant decrease was observed in phagocytic activity caused by smoking in female subjects. In other words, oxidative stress caused by smoking in male subjects may be involved in ROS production from neutrophils. Decreased phagocytic activity of neutrophils caused by smoking suggests that host defense functions were impaired in female subjects. A relationship between neutrophil functions and the amount of alcohol consumption was not observed. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes.

Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a 'hot spot' for horizontal gene transfer between microbes.     

Modulation of rat cecal microbiota by administration of raffinose and encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.     

Blood oxygenation using microbubble suspensions

Microbubbles have been used in a variety of fields and have unique properties, for example shrinking collapse, long lifetime, efficient gas solubility, a negatively charged surface, and the ability to produce free radicals. In medicine, microbubbles have been used mainly as diagnostic aids to scan various organs of the body, and they have recently been investigated for use in drug and gene delivery. However, there have been no reports of blood oxygenation by use of oxygen microbubble fluids without shell reagents. In this study, we demonstrated that nano or microbubbles can achieve oxygen supersaturation of fluids, and may be sufficiently small and safe for infusion into blood vessels. Although Po(2) increases in fluids resulting from use of microbubbles were inhibited by polar solvents, normal saline solution (NSS) was little affected. Thus, NSS is suitable for production of oxygen-rich fluid. In addition, oxygen microbubble NSS effectively improved hypoxic conditions in blood. Thus, use of oxygen microbubble (nanobubble) fluids is a potentially effective novel method for oxygenation of hypoxic tissues, for infection control, and for anticancer treatment.     

Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.     

Nerve Growth Factor as a Mitogen for a Pancreatic Carcinoid Cell Line

Abstract: Carcinoid tumors are a group of neuroendocrine neoplasms distributed widely throughout the body but most commonly occurring in the gut. These tumors retain many characteristics of their neural crest origin, including secretion of neuroactive peptides and responsiveness to neurotrophic substances. Nerve growth factor (NGF), a neurotrophic protein involved in maintenance and differentiation of peripheral sympathetic and sensory neurons, regulates growth of several neural tumor cells by inducing a differentiated phenotype and subsequent inhibition of cell growth rate. We examined the actions of NGF in a functioning human pancreatic carcinoid cell line (termed BON). NGF has no effect on the cytoarchitecture or constitutive secretion of bioamines in this carcinoid cell line. NGF, however, stimulates the in vitro cellular proliferation of BON cells. BON cells possess mRNA for the NGF receptors (p75^LNGFR and p140^trkA) and membrane-associated tyrosine kinase activity is increased in response to NGF. Both the mitogenic activity of NGF, as well as the receptor-linked tyrosine kinase activity, can be abrogated in BON cells by the trkA inhibitor K-252a and specific anti-NGF antibody. Our studies demonstrate that NGF is a mitogen for this carcinoid cell line without effect on cellular phenotype or cytoarchitecture. NGF may play a role in the development and progression of human carcinoid tumors.     

Biosynthesis of monosulfogangliotriaosylceramide and GM2 by N-acetylgalactosaminyltransferase from rat brain

Some properties of the enzyme activity that catalyzes the transfer of N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous lactosylceramide-II3-sulfate (SM3) and N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100,000 X g pellet of 6-day-old rat brain. The products from SM3 and GM3 were identified as gangliotriaosylceramide-II3-sulfate (SM2) and N-acetylneuraminosylgangliotriaosylceramide (GM2), respectively, by TLC-autoradiography. Optimal conditions for both activities were similar: pH (Hepes-NaOH), 7.0-7.5; detergent (heptylthioglucoside), 0.64% and Mn2+, 5-10 mM. The concentrations of the detergent optimal for both enzyme activities were also examined at various concentrations of the acceptors. The lower the amounts of acceptors, the less the amounts of detergent that were required, and vice versa, for the maximum activities. The acceptor-saturation curve for SM2 synthesis was triphasic, exhibiting a sigmoidal region at lower concentrations, a hyperbolic region and finally a descending region. For GM2 synthesis, the curve was biphasic without the descending region. The donor-saturation curves were classical hyperbolic ones for both syntheses. The Km values calculated for SM3 and GM3 were 0.37 and 0.19 mM, respectively, when the data corresponding to the hyperbolic regions were used for the double-reciprocal plots. The Km values for UDP-N-acetylgalactosamine in the SM2- and GM2-synthesis were 82 and 26 microM, respectively. SM3 and GM3 were the best acceptors for this enzyme preparation. From the results of the acceptor competition study, it was suggested that the two synthetic reactions are catalyzed by a single enzyme.     

Dose-dependent suppression of tea catechins with a galloyl moiety on postprandial hypertriglyceridemia in rats

Tea has long been believed to be a healthy beverage, and its beneficial effects are almost all attributed to catechins. The effect of catechins on postprandial hypertriglyceridemia in rats was investigated in this study. A lipid emulsion administered orally to rats with (-)-epigallocatechin gallate at a dose of 100 mg/kg resulted in the increase in plasma triacylglycerol being significantly inhibited after 1 and 2 h compared to the case without (-)-epigallocatechin gallate. The effect of (-)-epigallocatechin was weaker than that of (-)-epigallocatechin gallate. A tea extract (THEA-FLAN 90S), mainly composed of catechins with a galloyl moiety, dose-dependently suppressed postprandial triacylglycerol after the administration of a lipid emulsion at doses of 50-200 mg/kg. The administration of the tea extract alone at a dose of 200 mg/kg had no effect on the plasma triacylglycerol level. These results strongly suggest that catechins with a galloyl moiety would be promising agents for suppressing dietary fat absorption through the small intestine.     

Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.