Regulation of Inflammation by IL-17A and IL-17F Modulates Non-Alcoholic Fatty Liver Disease Pathogenesis

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. While it is well-accepted that inflammation is central to NAFLD pathogenesis, the immune pathway(s) orchestrating disease progression are poorly defined. Notably, IL-17RA signaling, via IL-17A, plays an important role in obesity-driven NAFLD pathogenesis. However, the role of the IL-17F, another IL-17RA ligand, in NAFLD pathogenesis has not been examined. Further, the cell types expressing IL-17RA and producing IL-17RA ligands in the pathogenesis of NAFLD have not been defined. Here, IL-17RA^-/-, IL-17A^-/-, IL-17F^-/- and wild-type (WT) mice were fed either standard chow diet or methionine and choline deficient diet (MCDD)—a diet known to induce steatosis and hepatic inflammation through beta-oxidation dysfunction—and hepatic inflammation and NAFLD progression were subsequently quantified. MCDD feeding augmented hepatic IL-17RA expression and significantly increased hepatic infiltration of macrophages and IL-17A and IL-17F producing CD4⁺ and CD8⁺ T cells in WT mice. In contrast, IL-17RA^-/-, IL-17A^-/-, and IL-17F^-/- mice, despite increased steatosis, exhibited significant protection from hepatocellular damage compared to WT controls. Protection from hepatocellular damage correlated with decreased levels of hepatic T-cell and macrophage infiltration and decreased expression of inflammatory mediators associated with NAFLD. In sum, our results indicate that the IL-17 axis also plays a role in a MCDD-induced model of NAFLD pathogenesis. Further, we show for the first time that IL-17F, and not only IL-17A, plays an important role in NAFLD driven inflammation.     

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

High and dry: post‐fire tree seedling establishment in subalpine forests decreases with post‐fire drought and large stand‐replacing burn patches

Aim Climate warming and increased wildfire activity are hypothesized to catalyse biogeographical shifts, reducing the resilience of fire‐prone forests world‐wide. Two key mechanisms underpinning hypotheses are: (1) reduced seed availability in large stand‐replacing burn patches, and (2) reduced seedling establishment/survival after post‐fire drought. We tested for regional evidence consistent with these mechanisms in an extensive fire‐prone forest biome by assessing post‐fire tree seedling establishment, a key indicator of forest resilience. Location Subalpine forests, US Rocky Mountains. Methods We analysed post‐fire tree seedling establishment from 184 field plots where stand‐replacing forest fires were followed by varying post‐fire climate conditions. Generalized linear mixed models tested how establishment rates varied with post‐fire drought severity and distance to seed source (among other relevant factors) for tree species with contrasting post‐fire regeneration adaptations. Results Total post‐fire tree seedling establishment (all species combined) declined sharply with greater post‐fire drought severity and with greater distance to seed sources (i.e. the interior of burn patches). Effects varied among key species groups. For conifers that dominate present‐day subalpine forests (Picea engelmannii, Abies lasiocarpa), post‐fire seedling establishment declined sharply with both factors. One exception was serotinous Pinus contorta, which did not vary with either factor. For montane species expected to move upslope under future climate change (Larix occidentalis, Pseudotsuga menziesii, Populus tremuloides) and upper treeline species (Pinus albicaulis), establishment was unrelated to either factor. Greater post‐fire tree seedling establishment on cooler/wetter aspects suggested local topographic refugia during post‐fire droughts. Main conclusions If future drought and wildfire patterns manifest as expected, post‐fire tree seedling establishment of species that currently characterize subalpine forests could be substantially reduced. Compensatory increases from lower montane and upper treeline species may partially offset these reductions, but our data suggest important near‐ to mid‐term shifts in the composition and structure of high‐elevation forests under continued climate warming and increased wildfire activity.     

Characterization and Cloning of ODAP Degrading Gene from a Soil Microbe

The strain BYT-1, capable of utilizing ODAP/DAP as a sole source of nitrogen and carbon was identified as Psuedomonas stutzeri by various microbial and biochemical tests. Transformation experiments showed that the ODAP utilizing property Is encoded by the plasmid. Restriction of plasmid DNA with Pstl, followed by cloning of fragments and screening of ODAP containing medium, led to the isolation of a clone with insert size of ?3.3 kb, which encoded ODAP metabolizing property. The growth and ODAP/DAP utilization by this clone (T_B) was almost similar to that of the wild type strain.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Solution and solid state behavior of Co2+, Ni2+ and Zn2+ tosylaminoacidate systems: Crystal and molecular structure of bis(N-tosylglycinato)tetraaquocobalt(II) and bis(N-tosyl-β-alaninato)tetraaquozinc(II) complexes

The interactions between N-tosylamino acids and cobalt(II), nickel(II) and zinc(II) ions in aqueous solution and in the solid state have been investigated. From concentrated aqueous solutions, compounds of general formula [M(II)(N-tosylaminoacidato)₂(H₂O)₄](M = Co(II), Ni(II) and N-tosylaminoacidato = N-tosylglycinate (Tsgly^?), N-tosyl-α- and -β-alaninate (Ts-α- and Ts-β-ala^?); M = Zn(II) and N-tosylaminoacidate = Tsgly^?, Ts-β-ala^?) and [Zn(II)(N- tosylaminoacidato)₂(H₂O)₂] were isolated and characterized by means of thermogravimetric, electronic and infrared spectra. For two of them: [Co(Tsgly)₂(H₂O)₄](I) and [Zn(Ts-β-ala)₂(H₂O)₄](II) the crystal and molecular structures were also determined. Both compounds crystallize in the monoclinic space group P2₁/c, with two formula units in a cell of dimensions: a = 13.007(6), b = 5.036(2), c = 18.925(7) Å, β = 102.33(3)° for (I) and a = 14.173(6), b = 5.469(2), c = 17.701(7) Å, β = 106.63(3)° for (II). The structures were solved by the heavy-atom method and refined by least-squares calculations to R = 0.031 and 0.064 for (I) and (II) respectively. The cobalt and zinc atoms lie in the centers of symmetry, each bonded to two amino- acid molecules through a carboxylic oxygen atom and four water molecules in a slightly tetragonally distorted octahedral geometry. The second carboxylic oxygen atom is not involved in metal coordination. Electronic and X ray-powder spectra suggest that the tetrahydrate complexes of Co²⁺, Ni²⁺ and Zn²⁺ ions of the same amino acids are isomorphous and isostructural. No coordinative interactions between ligand and metal ions were found in aqueous solution on varying the pH values before hydroxide precipitation.     

Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.