TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (“ricin-like”) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.     

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor. This designation, however, is controversial as a result of recent studies in which lysophosphatidylinositol (LPI) was identified as a GPR55 agonist. Defining a biological role for GPR55 requires GPR55 selective ligands that have been unavailable. From a β-arrestin, high-throughput, high-content screen of 300000 compounds run in collaboration with the Molecular Libraries Probe Production Centers Network initiative (PubChem AID1965), we identified potent GPR55 selective agonists. By modeling of the GPR55 activated state, we compared the GPR55 binding conformations of three of the novel agonists obtained from the screen, CID1792197, CID1172084, and CID2440433 (PubChem Compound IDs), with that of LPI. Our modeling indicates the molecular shapes and electrostatic potential distributions of these agonists mimic those of LPI; the GPR55 binding site accommodates ligands that have inverted-L or T shapes with long, thin profiles that can fit vertically deep in the receptor binding pocket while their broad head regions occupy a horizontal binding pocket near the GPR55 extracellular loops. Our results will allow the optimization and design of second-generation GPR55 ligands and provide a means for distinguishing GPR55 selective ligands from those interacting with cannabinoid receptors.     

The role of the individual Lhcas in photosystem I excitation energy trapping

In this work, we have investigated the role of the individual antenna complexes and of the low-energy forms in excitation energy transfer and trapping in Photosystem I of higher plants. To this aim, a series of Photosystem I (sub)complexes with different antenna size/composition/absorption have been studied by picosecond fluorescence spectroscopy. The data show that Lhca3 and Lhca4, which harbor the most red forms, have similar emission spectra (λ_max = 715–720 nm) and transfer excitation energy to the core with a relative slow rate of ∼25/ns. Differently, the energy transfer from Lhca1 and Lhca2, the “blue” antenna complexes, occurs about four times faster. In contrast to what is often assumed, it is shown that energy transfer from the Lhca1/4 and the Lhca2/3 dimer to the core occurs on a faster timescale than energy equilibration within these dimers. Furthermore, it is shown that all four monomers contribute almost equally to the transfer to the core and that the red forms slow down the overall trapping rate by about two times. Combining all the data allows the construction of a comprehensive picture of the excitation-energy transfer routes and rates in Photosystem I.     

Assessment of oxidant susceptibility of red blood cells in various species based on cell deformability

The present study was designed to investigate the oxidant susceptibility of red blood cells (RBC) from four species (echidna, human, koala, Tasmanian devil) based on changes in cellular deformability. These species were specifically chosen based on differences in lifestyle and/or biology associated with varied levels of oxidative stress. The major focus was the influence of superoxide radicals generated within the cell (phenazine methosulfate, PMS, 50 μM) or in the extracellular medium (xanthine oxidase-hypoxanthine, XO-HX, 0.1 U/ml XO) on RBC deformability at various shear stresses (SS). RBC deformability was assessed by laser-diffraction analysis using a "slit-flow ektacytometer". Both superoxide-generating treatments resulted in significant increases of methemoglobin for all species (p < 0.01), with Tasmanian devil RBC demonstrating the most sensitivity to either treatment. PMS caused impaired RBC deformability for all species, but vast interspecies variations were observed: human and koala cells exhibited a similar sigmoid-like response to SS, short-beaked echidna values were markedly lower and only increased slightly with SS, while Tasmanian devil RBC were extremely rigid. The effect of XO-HX on RBC deformability was less when compared with PMS (i.e., smaller increase in rigidity) with the exception of Tasmanian devil RBC which exhibited essentially no deformation even at the highest SS; Tasmanian devil RBC response to XO-HX was thus comparable to that observed with PMS. Our findings indicate that ektacytometry can be used to determine the oxidant susceptibility of RBC from different species which varies significantly among mammals representing diverse lifestyles and evolutionary histories. These differences in susceptibility are consistent with species-specific discrepancies between observed and allometrically-predicted life spans and are compatible with the oxidant theory of aging.     

Herbaceous production in South India—limiting factors and implications for large herbivores

This study’s goal was to better understand the growth pattern and limitations of the herbaceous production that supports South India’s rich large herbivore grazer assemblage. We conducted a fully factorial nitrogen and water (three levels each) treatment field experiment in the herbivore rich South Indian Western Ghats region to determine the seasonal pattern and the extent to which nitrogen and water availability limit herbaceous production. Graminoid production was found to be nitrogen limited. Despite low rainfall, additional water did not significantly increase overall biomass production nor extend growth in the dry season. Accumulated standing biomass was highest in the late wet season (November) and lowest in the dry season (May). Leaf nitrogen was highest in the early wet season (June) and lowest in the late dry season (March). Grazing had a positive effect on grass production by extending the growing season. Biomass production and graminoid leaf nitrogen concentration levels in the study area were similar to other tropical areas in the world. Also similar to other tropical large herbivore areas, the dry season poses an annual challenge for large herbivores in the study area —particularly the smaller bodied species—to satisfy their nutrient requirements.     

Preclinical evaluation of Som230 as a radiation mitigator in a mouse model: postexposure time window and mechanisms of action

The somatostatin analog SOM230 has potent radioprophylactic and radiation mitigating properties that are unrelated to cytoprotection but appear to be due to suppression of secretion of pancreatic enzymes into the intestinal lumen. To determine the maximal postirradiation time window for administration, male CD2F1 mice were exposed to 8.5-11 Gy total-body radiation; SOM230 (0.5, 2 or 5 mg/kg) or vehicle was given by twice daily subcutaneous injections for 14 days, beginning 24-72 h after irradiation, and 30-day animal survival was recorded. The contribution of the gut to systemic cytokine levels was estimated by analyzing plasma samples obtained simultaneously from the portal vein and carotid artery. The effect of SOM230 on cell trypsin secretion was assessed in vitro and intestinal proteolytic activity was measured in vivo. SOM230 was associated with a 40-60% absolute improvement in overall postirradiation survival when treatment was started 48 h after irradiation and even exhibited a statistically significant survival benefit when started at 72 h. SOM230 ameliorated the radiation-induced decrease in chemokine (C-X-C motif) ligand 9 (CXCL9). SOM230 inhibited pancreatic acinar cell trypsin secretion in vitro in a dose-dependent fashion and reduced intraluminal and intestinal tissue proteolytic activity in vivo. SOM230 is an excellent radiation mitigator with a postirradiation time window in excess of 48 h. The mechanism likely involves preservation of intestinal barrier function due to decreased secretion of pancreatic enzymes into the bowel lumen.     

SidL, an Aspergillus fumigatus transacetylase involved in biosynthesis of the siderophores ferricrocin and hydroxyferricrocin

The opportunistic fungal pathogen Aspergillus fumigatus produces four types of siderophores, low-molecular-mass iron chelators: it excretes fusarinine C (FsC) and triacetylfusarinine C (TAFC) for iron uptake and accumulates ferricrocin (FC) for hyphal and hydroxyferricrocin (HFC) for conidial iron distribution and storage. Siderophore biosynthesis has recently been shown to be crucial for fungal virulence. Here we identified a new component of the fungal siderophore biosynthetic machinery: AFUA_1G04450, termed SidL. SidL is conserved only in siderophore-producing ascomycetes and shows similarity to transacylases involved in bacterial siderophore biosynthesis and the N(5)-hydroxyornithine:anhydromevalonyl coenzyme A-N(5)-transacylase SidF, which is essential for TAFC biosynthesis. Inactivation of SidL in A. fumigatus decreased FC biosynthesis during iron starvation and completely blocked FC biosynthesis during iron-replete growth. In agreement with these findings, SidL deficiency blocked conidial accumulation of FC-derived HFC under iron-replete conditions, which delayed germination and decreased the size of conidia and their resistance to oxidative stress. Remarkably, the sidL gene is not clustered with other siderophore-biosynthetic genes, and its expression is not affected by iron availability. Tagging of SidL with enhanced green fluorescent protein suggested a cytosolic localization of the FC-biosynthetic machinery. Taken together, these data suggest that SidL is a constitutively active N(5)-hydroxyornithine-acetylase required for FC biosynthesis, in particular under iron-replete conditions. Moreover, this study revealed the unexpected complexity of siderophore biosynthesis, indicating the existence of an additional, iron-repressed N(5)-hydroxyornithine-acetylase.     

Energy advantages of orientation to solar radiation in three African ruminants

Animal orientation relative to incident solar radiation allows an animal to effectively adjust the amount of radiant heat gained from an environment. Yet recent literature found ruminants to primarily orientate north/south and proposed magnetic alignment as the most parsimonious explanation. To test whether such northerly orientation has an energy advantage, we used heated cylindrical models to estimate energy costs of thermoregulation associated with north and east orientations of three species of African ruminants under cool winter conditions. Concurrent behavioural observations revealed that eland, blue wildebeest and impala did not preferentially orientate north/south during warm summer or cool winter conditions. Instead, all three species preferred to orientate perpendicular to incident solar radiation during winter and parallel to incident solar radiation during summer, throughout the day. On clear winter days with little wind, more than 60% of animal orientation preference could be accounted for by the energy savings associated with that orientation. Thus energy demands are likely to be the primary driver of animal orientation preferences.     

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10¹ to 10² bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.