The stiffness‐controlled release of interleukin‐6 by cardiac fibroblasts is dependent on integrin α2β1

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin‐6 (IL‐6), interleukin‐11 (IL‐11) and soluble receptor of IL‐6 (sIL‐6R). It also examines the roles of integrin α2β1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2β1 integrin count and released higher levels of IL‐6 and sIL‐6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL‐11 content. The silencing of the α2 integrin subunit decreased the release of IL‐6. Similar effects were induced by TC‐I 15 (an α2β1 integrin inhibitor). The IL‐6 levels in the serum and heart were markedly lower in α2 integrin‐deficient mice B6.Cg‐Itga2^{tm1.1Tkun/tm1.1Tkun} than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL‐6 level. sIL‐6R secretion is not dependent on α2β1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL‐6 by cardiac fibroblasts, and this effect is dependent on α2β1 integrin and kinase Src activation.     

Apparent Precaecal Digestibility of Nutrients and Level of Endogenous Nitrogen in Digesta of the Small Intestine of Growing Pigs as Affected by Various Digesta Viscosities

Sixteen male growing pigs of about 24kg BW were fitted with both a duodenal re-entrant and a post-valve T-shaped cannula inserted in the caecum. The animals were divided into four groups. Each group received one of the following diets: corn starch-soybean protein isolate-based diet without (diet C) and with carboxymethylcellulose (diet CMC) or a rye-wheat-based diet without (diet RW) and with xylanase addition (diet RWX). The diets provided similar levels of apparent precaecal digestible crude protein (CP), lysine, methionine+cystine, threonine and tryptophan. Additionally, [¹⁵N]-yeast was given with the diets during the first 10 days of the experiment. For estimation of digesta viscosity, N-flow of dietary and endogenous origin, apparent precaecal digestibilities of dry matter (DM), CP, amino acids and non starch polysaccharides (NSP) (only in pigs fed diets RW and RWX), ileal and duodenal digesta were quantitatively collected on day 16 and 17, respectively. The endogenous N-proportion was measured by the ratio of ¹⁵N enrichment in the digesta and urine. The duodenal and ileal digesta supernatant viscosity increased as carboxymethylcellulose was included into the diet. Xylanase addition to the rye-wheat based diet reduced the viscosity in the ileal digesta. There were no differences in precaecal digestibilities of DM, CP and amino acids between diet C and CMC. The precaecal digestibilities of DM and soluble and insoluble NSP increased from 69.5% to 73.9%, from 1.3% to 47.9% and from 17.0% to 35.4%, respectively, as xylanase was added to the rye-wheat-based diet. The apparent precaecal digestibility of most essential amino acids increased by 2 to 5 percent units. The amounts of endogenous N at the duodenal level were estimated to be 158, 233, 313 and 276mg per 12h per kg^0.75 BW of pigs fed diets C, CMC, RW and RWX, respectively. The corresponding values at the ileal level were 95, 107, 164 and 150mg per 12h per kg^0.75 BW. For endogenous N amounts, significant differences were observed between diets C and CMC (duodenum) and also between semi-purified and cereal-based diets (duodenum and ileum). Methodological aspects for the estimation of endogenous N using the isotope dilution technique are discussed. Obviously, the digesta viscosity per se does not affect the nutrient absorption and endogenous N flow within the small intestine of pigs. Other properties of complex dietary fibre, digesta passage rate or bacterial activity probably contribute to the observed changes.     

The thylakoid protease Deg2 is involved in stress-related degradation of the photosystem II light-harvesting protein Lhcb6 in Arabidopsis thaliana

? The thylakoid protease Deg2 is a serine-type protease peripherally attached to the stromal side of the thylakoid membrane. Given the lack of knowledge concerning its function, two T-DNA insertion lines devoid of Deg2 were prepared to study the functional importance of this protease in Arabidopsis thaliana. ? The phenotypic appearance of deg2 mutants was studied using a combination of stereo and transmission electron microscopy, and short-stress-mediated degradation of apoproteins of minor light-harvesting antennae of photosystem II (PSII) was analysed by immunoblotting in the mutants in comparison with wild-type plants. ? Deg2 repression produced a phenotype in which reduced leaf area and modified chloroplast ultrastructure of older leaves were the most prominent features. In contrast to the wild type, the chloroplasts of second-whorl leaves of 4-wk-old deg2 mutants did not display features typical of the early senescence phase, such as undulation of the chloroplast envelope and thylakoids. The ability to degrade the photosystem II light-harvesting protein Lhcb6 apoprotein in response to brief high-salt, wounding, high-temperature and high-irradiance stress was demonstrated to be impaired in deg2 mutants. ? Our results suggest that Deg2 is required for normal plant development, including the chloroplast life cycle, and has an important function in the degradation of Lhcb6 in response to short-duration stresses.     

Influence of Trace Elements on Stabilization of Aqueous Solutions of Ascorbic Acid

Together with vitamin C, zinc, selenium, manganese, and magnesium play a vital role in the preservation of organs scheduled for transplantation. In the present study, it is shown that addition of 1?mg/l of these elements influences the stability of 0.3?mM ascorbic acid solutions. The solution??s stability was estimated using an accelerated stability test. The concentration of vitamin C was measured using a validated spectrophotometric method, which uses the reduction of 2,6-dichlorophenoloindophenol by ascorbic acid. Elevated temperatures, the factor accelerating substances?? decomposition reaction rate, were used in the tests. The research was conducted at two temperatures at intervals of 10?°C: 80?±?0.1 and 90?±?0.1?°C. It was stated that the studied substances?? decomposition occurred in accordance with the equation for first-order reactions. The function of the logarithmic concentration (log%C) over time was revealed to be rectilinear. This dependence was used to determine the kinetics of decomposition reaction rate parameters. The stabilization of vitamin C solutions was measured as the time in which 10?% of the substance decomposed at 20 and 0?°C. Addition of Se(IV) or Mg(II) ions significantly increase the stability of ascorbic acid solution (??34 and ??16?%, respectively), but Zn(II) causes a significant decrease in stability by ??23?%. Addition of Mn(II) has no significant influence on vitamin C stability.     

The mechanism of 1,2,3,4-tetrahydroisoquinolines neuroprotection: the importance of free radicals scavenging properties and inhibition of glutamate-induced excitotoxicity

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4-tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free-radical generation in an abiotic system, as well as indices of neurotoxicity (caspase-3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7-day-old rats, 1MeTIQ prevented the glutamate-induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK-801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate-induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.     

Patching of ganglioside(M1) in human erythrocytes - distribution of CD47 and CD59 in patched and curved membrane

Membrane rafts may act as platforms for membrane protein signalling. Rafts have also been implicated in the sorting of membrane components during membrane budding. We have studied by fluorescence microscopy cross-linking of ganglioside GM1 in the human erythrocyte membrane, and how membrane proteins CD47 and CD59 distribute in GM1 patched discoid cells and calcium-induced echinocytic cells. Patching of ganglioside(M1) (GM1) by cholera toxin subunit B (CTB) plus anti-CTB resulted in the formation of usually 40-60 GM1 patches distributed over the membrane in discoid erythrocytes. Pre-treatment of erythrocytes with methyl-beta-cyclodextrin abolished GM1 patching. GM1 patching was insensitive to pre-fixation (paraformaldehyde) of cells. Patching of GM1 did not affect the discoid shape of erythrocytes. Membrane proteins CD47 and CD59 did not accumulate into GM1 patches. No capping of patches occurred. GM1 accumulated in calcium-induced echinocytic spiculae. Also CD59, but not CD47, accumulated in spiculae. However, CD59 showed a low degree of co-localization with GM1 and frequently accumulated in different spiculae than GM1. In conclusion, our study describes a novel method for examining properties and composition of rafts. The study characterizes raft patching in the human erythrocyte membrane and emphasizes the mobility and 'echinophilicity' of GM1. Glycosyl phosphatidylinositol-anchored CD59 was identified as a mobile 'echinophilic' but 'raftophobic(GM1)' protein. Largely immobile CD47 showed no segregation.     

New evidence for the use of root foods in pre-agrarian subsistence recovered from the late Mesolithic site at Halsskov, Denmark

Roots and tubers, together with other plant storage organs such as rhizomes, bulbs, corms, etc. are known to be a source of human food. Until very recently, however, remains of root foods were rarely identified from archaeological contexts in temperate Europe. New evidence for the use of root foods has been recently recovered from the early Erteb?lle settlement at Halsskov in Denmark. Remains included charred bulbs of Allium cf. ursinum and tubers of Conopodium majus. They were accompanied by charred remains of parenchymatous tissues derived from underground parts of other plants. All parenchymatous remains were recovered from features that could be interpreted as pit-cooking depressions. A minimum of two periods of root food gathering can be assumed, spring to early summer and autumn. The presence of charred seeds of Nuphar pumilum suggests that either the seeds or rhizomes (or both) were use as food. Received September 4, 2001 / Accepted February 27, 2002     

Genetic diversity and relatedness within packs in an intensely hunted population of wolvesCanis lupus

A population of grey wolvesCanis lupus Linnaeus, 1758 inhabiting Bia?owie?a Primeval Forest (BPF) on the Polish-Belarussian border has recovered after near extermination in the 1970s. Currently, it is intensively hunted in the Belarussian part of BPF and protected in the Polish part. We used a combination of molecular analysis, radiotracking, and field observation to study genetic diversity of the population after natural recolonisation and the consequences of heavy hunting for the genetic composition and social structure of wolf packs. Both microsatellite and mtDNA analyses revealed high genetic diversity. For 29 individuals and 20 microsatellite loci, the mean expected heterozygosity was 0.733. Four mtDNA haplotypes were found. Three of them had earlier been described from Europe. Their geographic distribution suggests that wolves recolonising BPF immigrated mainly from the north-east, and less effectively from the east and south-east. We traced the composition of 6 packs for a total of 26 pack-years. Packs were family units (a breeding pair with offspring) with occasional adoption of unrelated adult males, which occurred more frequently in packs living in the Belarussian part of the BPF, due to heavy hunting and poaching. Breeding pairs were half-sibs or unrelated wolves. Pair-bonds in the breeding pair lasted from 1 to 4 years and usually broke by the death of one or both mates. Successors of breeding females were their daughters, while a successor of a breeding male could be either his son or an alien wolf. As is evident from Bia?owie?a’s wolves, high genetic diversity may result from immigration of outside individuals, which are easily recruited to a heavily exploited local population.