Genistein, a plant-derived isoflavone, counteracts the antilipolytic action of insulin in isolated rat adipocytes

Genistein is a phytoestrogen exerting numerous biological effects. Its direct influence on adipocyte metabolism and leptin secretion was previously demonstrated. This study aimed to determine whether genistein antagonizes the antilipolytic action of insulin in rat adipocytes. Freshly isolated adipose cells were incubated for 90 min with epinephrine, epinephrine with insulin and epinephrine with a specific inhibitor of protein kinase A (H-89) at different concentrations of genistein (0, 6.25, 12.5, 25, 50 and 100 μM). Genistein failed to affect epinephrine-induced glycerol release, however, the inhibitory action of insulin on epinephrine-induced lipolysis was significantly abrogated in cells exposed to the phytoestrogen (12.5–100 μM). The increase in insulin concentration did not suppress the genistein effect. Its inhibitory influence on the antilipolytic action of insulin was accompanied by a substantial rise in cAMP in adipocytes. This rise appeared despite the presence of 10 nM insulin in the incubation medium. Further experiments, in which insulin was replaced by H-89, revealed that the antilipolytic action of protein kinase A inhibitor on epinephrine-induced lipolysis was not affected by genistein. This means that genistein counteracted the antilipolytic action of insulin due to the increase in cAMP levels and activation of protein kinase A in adipocytes. The observed attenuation of the inhibitory effect of insulin on triglyceride breakdown evoked by genistein was not related to its estrogenic activities, as evidenced in experiments employing the intracellular estrogen receptor blocker, ICI 182,780. Moreover, it was found that genistein-induced impairment of the antilipolytic action of insulin was not accompanied by changes in the proportion between fatty acids and glycerol released from adipocytes. The ability of genistein to counteract the antilipolytic action of insulin may contribute to the decreased triglyceride accumulation in adipose tissue.     

Denitrification with endogenous carbon source at low C/N and its effect on P(3HB) accumulation

The objective of the work reported here was to determine whether the ratio of COD/Nox has an impact on poly-beta-hydroxybutyrate (PHB) metabolism in activated sludge. Furthermore, it was tested if the ratio influenced the percentage use of organic compounds present in wastewater, for endogenous respiration, oxidation, accumulation and denitrification. Gas flow rate in SBR reactor was controlled by thermal mass flow controller (TMFC). Constant amount of air entering sequencing batch reactor was automatically adjusted to stable set-point 2mg O2 L(-1). It means that DO concentration in the reactor could change with oxygen uptake. During the filling period and part of the reaction time DO was nearly zero. Feast period of the external substrate availability and famine period of little amount or no external carbon availability were determined. At 23 h of the reaction time, and COD/Nox ratio 8, denitrification took place only during feast period. What was interesting, poly-beta-hydroxybutyrate degradation was observed in the feast period as well. However, at 11h of the reaction time and COD/Nox ratio 37, denitrification occurred in feast and famine period. In the feast period PHB was accumulated and in the famine period was used as the endogenous carbon source. COD consumption to reduce 1mg N-nitrate was ranging from 1.15 to 6.26 depending on carbon source and increased when exogenous and endogenous carbon were used by activated sludge. The increase in PHB content from 0.25 to 0.43 Cmol/Cmol resulted in a double increase in the amount of nitrogen removed due to denitrification was observed.     

Calcium- and pH-dependent localization of annexin A6 isoforms in Balb/3T3 fibroblasts reflecting their potential participation in vesicular transport

Annexin A6 (AnxA6), calcium- and membrane-binding protein, is involved in membrane dynamics. It exists in the cell in two isoforms, AnxA6-1 and AnxA6-2, varying only by the VAAEIL sequence. In most cells, AnxA6-1 predominates. A limited number of observations suggests that both isoforms differ from each other functionally. The EGF-dependent Ca(2+) influx in A431 cells is inhibited only by AnxA6-1. Moreover, AnxA6-2 was found to exhibit higher affinity for Ca(2+). In this report we addressed the potential significance of the VAAEIL deletion in AnxA6-2. For this purpose, we expressed AnxA6 isoform cDNAs in bacteria or mouse Balb/3T3 fibroblasts. The recombinant AnxA6-2 was characterized by a less extended molecular shape than that of AnxA6-1 and required a narrower [Ca(2+)] range to bind liposomes. Upon lowering pH in the presence of EGTA recombinant AnxA6-2 became less hydrophobic than AnxA6-1 as revealed by the Triton X-114 partition. Furthermore, AnxA6-2 revealed stronger F-actin binding than that of AnxA6-1. Immunofluorescence microscopy showed that the EGFP-tagged AnxA6 isoforms expressed in Balb/3T3 fibroblasts relocate in a Ca(2+)- and H(+)-sensitive manner to the vesicular structures in a perinuclear region or in cytosol. Cell fractionation showed that in resting conditions AnxA6-1 is associated with early endosomes and AnxA6-2 with late endosomes, and an increase in [Ca(2+)] and/or [H(+)] induced their opposite distribution. These findings suggest a potentially independent regulation, localization, and function of AnxA6 isoforms in Balb/3T3 fibroblasts. More generally, our findings suggest distinct functions of AnxA6 isoforms in membrane dynamics.     

Plant-derived EpCAM antigen induces protective anti-cancer response

Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.     

Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily,but exhibits an unusual active site

Background  

Catalytic domains of Type II restriction endonucleases (REases) belong to a few unrelated three-dimensional folds. While the PD-(D/E)XK fold is most common among these enzymes, crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI). Bioinformatics analyses supported by mutagenesis experiments suggested that some REases belong to the HNH fold (e.g. R.KpnI), and that a small group represented by R.Eco29kI belongs to the GIY-YIG fold. However, for a large fraction of REases with known sequences, the three-dimensional fold and the architecture of the active site remain unknown, mostly due to extreme sequence divergence that hampers detection of homology to enzymes with known folds.     

A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus

Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.     

Cell death upon epigenetic genome methylation: a novel function of methyl-specific deoxyribonucleases

Background  

Alteration in epigenetic methylation can affect gene expression and other processes. In Prokaryota, DNA methyltransferase genes frequently move between genomes and present a potential threat. A methyl-specific deoxyribonuclease, McrBC, of Escherichia coli cuts invading methylated DNAs. Here we examined whether McrBC competes with genome methylation systems through host killing by chromosome cleavage.     

Interactions between phytoplankton and zooplankton in the hypertrophic Swarzedzkie Lake in western Poland

The hypertrophic Swarzdzkie Lake, Poland, is characterized byhigh species diversity, abundance and biomass of both phytoplanktonand zooplankton (up to 99.5 mg WW L^–1 and 817.75 µgDW L^–1, respectively). The community grazing rate calculatedwith the use of two empirical models, and based on herbivorouscrustaceans, peaked in spring and early autumn up to 150.6%of water filtered per day, and was the lowest during winter.Simple statistics revealed a positive correlation between zooplanktonbiomass and chlorophyll a concentration (r = 0.404, P = 0.033)and between zooplankton abundance and phytoplankton biomass(r = 0.42, P = 0.028). Canonical statistics indicated, however,that the relationship exists only with size groups and/or livingforms of a few taxonomical groups of phytoplankton. Redundancyanalysis (RDA) confirmed a positive influence of the communitygrazing rate on micro- and nanoplanktonic Cryptophyceae, butnot on the microplanktonic Cyanobacteria, as was suggested bycanonical correlation analysis. RDA also indicated a weak negativeinfluence on nanoplanktonic Euglenophyceae and Chlorophyceaeexerted by filtering crustaceans. Some taxonomically diverseflagellated nanoplanktonic algae were grazing sensitive, whereasmicroplanktonic cryptophytes and coenobial green algae weresignificantly grazing resistant.     

Magnitude of mesozooplankton variability: a case study from the Marginal Ice Zone of the Barents Sea in spring

Zooplankton was studied on eight stations in the marginal icezone (MIZ) of the Barents Sea, in May 1999, along two transectsacross the ice edge. On each station, physical background measurementsand zooplankton samples were taken every 6 h over a 24 h periodat five discrete depth intervals. Cluster analysis revealedseparation of open water stations from all ice stations as wellas high similarity level among replicates belonging to particularstation. Based on five replicates per station, analysis of variance(ANOVA) confirmed significant differences (P < 0.05) in abundancesof the main mesozooplankton taxa among stations. Relations betweenthe zooplankton community and environmental parameters wereestablished using redundancy analysis (CANOCO). In total, 55%of mesozooplankton variability within studied area was explainedby eight variables with significant conditional effects: depthstratum, fluorescence, temperature, salinity, bottom depth,latitude, bloom situation, and ice concentration. GLM modelssupported supposition about clear and negative relationshipbetween concentration of Oithona similis, and overall mesozooplanktondiversity. The analyses showed a dynamic relationship betweenmesozooplankton distribution and hydrological conditions onshort-term scale. Furthermore, our study demonstrated that variabilityin the physical environment of dynamic MIZ of the Barents Seahas measurable effect on the Arctic pelagic ecosystem.