Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.     

Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase

We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.     

Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, 17beta-estradiol and testosterone as well as their sulfate esters. These brain neurosteroids are synthesized by cytochrome P450s (P450scc, P45017alpha and P450arom) from endogenous cholesterol. Synthesis is acutely dependent on the Ca(2+) influx attendant upon neuron-neuron communication via N-methyl-D-aspartate (NMDA) receptors. Pregnenolone sulfate, estradiol and corticosterone rapidly modulate neuronal signal transduction and the induction of long-term potentiation via NMDA receptors and putative membrane steroid receptors. Brain neurosteroids are therefore promising neuromodulators that may either activate or inactivate neuron-neuron communication, thereby mediating learning and memory in the hippocampus.     

Endosomal/lysosomal targeting of a single helper T-cell epitope of an intracellular bacterium by DNA immunisation induces a specific T-cell subset and partial protective immunity in vivo

We evaluated here the effect of the intracellular targeting of a helper T-cell (Th) epitope, literiolysin O 215-226 derived from Listeria monocytogenes, on induction of a specific Th by gene gun immunisation. Immunisation of C3H/He mice with pE215LAMP plasmid encoding the Th epitope fused with the endosomal/lysosomal targeting signal of lysosome-associated membrane protein (LAMP)-1 gave the epitope-specific proliferative responses of CD4(+) T lymphocytes. In addition, specific interferon-gamma production from the splenocytes was observed. Concomitantly, pE215LAMP-immunised mice showed moderate, but significant protective immunity against listerial challenge. These results suggest that the intracellular targeting of a Th epitope to endosomal/lysosomal compartments by DNA immunisation is useful for eliciting a specific Th subset in vivo.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min⁻¹  mg protein⁻¹ . The apparent K _m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

     

Enhancement of Inflammatory Protein Expression and Nuclear Factor Κb (NF-Κb) Activity by Trichostatin A (TSA) in OP9 Preadipocytes

The production of inflammatory proteins such as interleukin-6 (IL-6) by preadipocytes and mature adipocytes is closely associated with the impairment of systemic glucose homeostasis. However, precisely how the production is regulated and the roles of histone deacetylases (HDACs) remain largely unknown. The aim of this study was to establish whether HDAC inhibitors affect the expression of inflammatory proteins in pre/mature adipocytes, and, if so, to determine the mechanism involved. Trichostatin A (TSA), an HDAC inhibitor, enhanced lipopolysaccharide (LPS)-induced production of IL-6 in OP9 preadipocytes but not the mature adipocytes. Moreover, TSA also enhanced palmitic acid-induced IL-6 production and the expression of inflammatory genes induced by LPS in preadipocytes. Although TSA did not affect TLR4 mRNA expression or the activation of MAPKs, a reporter gene assay revealed that the LPS-induced increase in nuclear factor κB (NF-κB) activity was enhanced by TSA. Moreover, TSA increased the level of NF-κB p65 acetylation at lysine 310 and duration of its translocation into the nucleus, which leads to enhancement of NF-κB activity and subsequently expression of inflammatory genes. These findings shed new light on the regulatory roles of HDACs in preadipocytes in the production of inflammatory proteins.     

DNA topoisomerase II interacts with Lim15/Dmc1 in meiosis

Lim15/Dmc1 is a meiosis specific RecA-like protein. Here we propose its participation in meiotic chromosome pairing-related events along with DNA topoisomerase II. Analysis of protein–protein interactions using in vitro binding assays provided evidence that Coprinus cinereus DNA topoisomerase II (CcTopII) specifically interacts with C.cinereus Lim15/Dmc1 (CcLim15). Co-immunoprecipitation experiments also indicated that the CcLim15 protein interacts with CcTopII in vivo. Furthermore, a significant proportion of CcLim15 and CcTopII could be shown to co-localize on chromosomes from the leptotene to the zygotene stage. Interestingly, CcLim15 can potently activate the relaxation/catenation activity of CcTopII in vitro, and CcTopII suppresses CcLim15-dependent strand transfer activity. On the other hand, while enhancement of CcLim15's DNA-dependent ATPase activity by CcTopII was found in vitro, the same enzyme activity of CcTopII was inhibited by adding CcLim15. The interaction of CcLim15 and CcTopII may facilitate pairing of homologous chromosomes.     

IFN-gamma overcomes low responsiveness of myeloid dendritic cells to CpG DNA

Dendritic cells (DC) are professional APC that have an extraordinary capacity to prime naive T cells. It has been reported that human DC subsets express distinct toll-like receptor (TLR), which influences their function. In mice, we observed that plasmocytoid DC (pDC) express a higher level of TLR9 compared with myeloid DC (mDC) cultured with GM-CSF. However, we demonstrated that stimulation with IFN-gamma is capable of upregulating TLR9 expression in mDC to a level comparable with expression in pDC. Consistent with this observation, IL-12 p40 and IL-6 mRNA expression and IL-12 p70 secretion in response to CpG-oligodeoxynucleotides are enhanced in mDC pretreated with IFN-gamma compared with untreated cells. Therefore, TLR-mediated responses of DC subsets may be influenced not only by signals delivered by pathogens but also by regulatory signals from cytokines such as IFN-gamma.     

Effect of estrogenic activity, and phytoestrogen and organochlorine pesticide contents in an experimental fish diet on reproduction and hepatic vitellogenin production in medaka (Oryzias latipes)

Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen beta-receptors than estrogen alpha-receptors. Organochlorine pesticides such as hexachlorobenzene, beta-benzene hexachloride (BHC), and gamma-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 +/- 0.6 microg/g; daidzein, 37.3 +/- 0.2 microg/g) for 28 days compared with those fed diet A (genistein, < 0.8 microg/g; daidzein, < 0.8 microg/g) or diet B (genistein, 1.4 +/- 0.1 microg/g; daidzein, 2.0 +/- 0.1 microg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet.