PTPN22 1858C>T gene polymorphism in patients with SLE: association with serological and clinical results

To assess the association between PTPN22 1858C>T gene polymorphism and susceptibility to, and clinical presentation of, systemic lupus erythematosus (SLE). Our study included 135 SLE patients (120 women and 15 men; mean age 45.1 years; mean course of disease from 0.5 to 31 years) and 201 healthy subjects. The PTPN22 1858C>T gene polymorphism was genotyped by polymerase chain reaction restriction fragment length polymorphism. A significantly higher incidence of genotype CT in patients with SLE (36.3 %) was found, compared with the control group (24.9 %). The frequencies of C1858 and T1858 alleles were 78.1 and 21.9 % in SLE patients and 86.1 and 13.9 % in controls, respectively. Significantly higher SLE susceptibility was observed in patients carrying at least one T allele (p = 0.009; OR 1.86; 95 % CI 0.14–3.05). Significant association of the PTPN22 T1858 allele (CT + TT vs.CC) and secondary antiphospholipid syndrome was observed (p = 0.049). In SLE patients carrying the T1858 allele, higher levels of antiphospholipid antibodies (anticardiolipin antibodies and/or lupus anticoagulant) were found (p = 0.030; OR 2.17; 95 % CI 1.07–4.44).     

Matrix remodeling stimulates stromal autophagy, “fueling” cancer cell mitochondrial metabolism and metastasis

We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1^-/- null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1^-/- MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy (“self-eating”) and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the “Autophagic Tumor Stroma Model of Cancer” and identifies a novel “extracellular matrix”-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism.     

Active polyphenolic compounds,nutrient contents and antioxidant capacity of extruded fish feed containing purple coneflower (Echinacea purpurea (L.) Moench.)

The growth of fish is directly dependent on feed composition and quality. Medicinal plants can be added to fish feed as adjuvant therapy for the prevention of fish diseases. The purple coneflower (Echinacea purpurea (L.) Moench.) has been reported to have multiple biological effects, including immunomodulatory and antioxidant activity. The most active compounds of E. purpurea are polyphenols - caffeic acid derivatives: caftaric acid, chlorogenic acid, cynarin, echinacoside and cichoric acid.Due to a relatively limited number of studies on the use of the purple coneflower as a nutritional supplement for fish feeding, extruded fish feed with addition of Echinacea roots was produced. In the feed total phenolic content, selected polyphenol contents, the energetic value, nutrient contents and antioxidant capacity were examined.The results indicate that fish feed with addition of the Echinacea has a great potential to be a good source of natural radical scavengers, for example polyphenols, and nutritive ingredients. Antioxidant properties of feed were well correlated with the coneflower content. The study findings confirmed that high-temperature extrusion-cooking process does not deactivate phenolic antioxidant compounds, which are present both in the Echinacea roots and in the final product. Fish feed with addition of E. purpurea can be used as a nutritional supplement in the prevention of fish diseases caused by oxidative stress.     

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O57 containing an amide of D-galacturonic acid with L-alanine

The O-polysaccharide (O-antigen) was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O57:H29. Studies by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments, showed that the polysaccharide contains an amide of D-galacturonic acid with L-alanine and has the following pentasaccharide repeating unit: [formula: see text]     

Biochemical and immunohistochemical study on physiological activity and distribution of hepatic cathepsin D

Cathepsin D (EC 3.4.23.5) is a lysosomal endopeptidase physiologically present at very low concentration in different tissues. The aim of the study was to estimate the physiological activity and distribution of cathepsin D in the liver. Four groups of ten-week-old male Wistar rats were raised without xenobiotics and sacrificed on day 4, 42, 47 and 84 of the experiment, and their livers were taken for immunohistochemical and biochemical investigation. Immunostaining for cathepsin D was evaluated by light microscope. Activity of the free and bound fractions of hepatic cathepsin D was measured spectrophotometrically. Immunohistochemical staining for cathepsin D was positive in Browicz-Kupffer cells in some but not in all rat liver specimens of each experimental group. The staining pattern was cytoplasmic and granular. Occasionally the positive stained endothelial cells were also found. No activity of cathepsin D in hepatocytes was detected. The positive immunostaining was found in livers with high enzyme activity in the biochemical investigation. No significant differences in activity of the free and bound fractions of cathepsin D among the different age groups were noted. However, the higher, age-dependent activity (p>0.05) of the free fraction was observed in the youngest and the two-middle groups of rats that were sacrificed on day 42 and 47 than in the oldest one. The bound fraction did not reveal such changes. It could be concluded that there were no differences in the activity of hepatic free and bound fractions of cathepsin D in male Wistar rats of various reproductive age. The rat Browicz-Kupffer cells revealed the highest activity of cathepsin D.     

Oenothein B's contribution to the anti-inflammatory and antioxidant activity of Epilobium sp

Willow herb tea or preparation are available and relatively popular in the European market, and claimed to be effective inter alia because of their anti-inflammatory activity. The present study is therefore aimed at comparing the anti-inflammatory and antioxidant activity of extracts of the three most popular Epilobium species (E. angustifolium, E. hirsutum and E. parviflorum) and at juxtaposing this activity against the dominating compounds from the following extracts: oenothein B (OeB), quercetin-3-O-glucuronide and myricetin-3-O-rhamnoside. The phytochemical analysis of the extracts has shown that OeB quantities vary between 20% and 35%, while flavonoids content does not exceed 2%. All extracts have inhibited the activity of hyaluronidase and lipoxygenase with IC₅₀ around 5 μg/ml and 25 μg/ml. The inhibition of hyaluronidase is related with the presence of OeB, a strong inhibitor of this enzyme (IC₅₀ 1.1 μM). Additionally, the extracts inhibited myeloperoxidase (MPO) release from stimulated neutrophils. OeB inhibited MPO release similarly to the anti-inflammatory drug indomethacin with IC₅₀ 7.7 μM and 15.4 μM, respectively. Tested extracts significantly reduced the production of reactive oxygen species (ROS) from f-MLP and PMA induced neutrophils with IC₅₀ 5 μg/ml and 25 μg/ml, respectively. The flavonoids content seems to exert little influence on extracts’ activity, contrary to OeB, whose high concentration explains the activity of extract obtained from Epilobium. Tested currently marketed Epilobium preparations are often wrongly assigned, but we should stress that the level of OeB in all tested herbs was high and always exceeded 2% in raw material.     

5‐Azacytydine and resveratrol reverse senescence and ageing of adipose stem cells via modulation of mitochondrial dynamics and autophagy

Obesity and endocrine disorders have become prevalent issues in the field of both human and veterinary medicine. Equine metabolic syndrome is a complex disorder involving alternation in metabolism and chronic systemic inflammation. It has been shown that unfavourable microenvironment of inflamed adipose tissue negatively affects adipose stem cell population (ASC) residing within, markedly limiting their therapeutic potential. ASCs_EMS are characterized by increased senescence apoptosis, excessive accumulation of reactive oxygen species (ROS), mitochondria deterioration and “autophagic flux.” The aim of the present study was to evaluate whether treatment of ASCs_EMS with a combination of 5‐azacytydine (AZA) and resveratrol (RES) would reverse aged phenotype of these cells. For this reason, we performed the following analyzes: molecular biology (RT‐PCR), microscopic (immunofluorescence, TEM) and flow cytometry (JC‐1, ROS, Ki67). We evaluated the mitochondrial status, dynamics and clearance as well as autophagic pathways. Furthermore, we investigated epigenetic alternations in treated cells by measuring the expression of TET genes and analysis of DNA methylation status. We have demonstrated that AZA/RES treatment of ASCs_EMS is able to rejuvenate these cells by modulating mitochondrial dynamics, in particular by promoting mitochondrial fusion over fission. After AZA/RES treatment, ASCs_EMS were characterized by increased proliferation rate, decreased apoptosis and senescence and lower ROS accumulation. Our findings offer a novel approach and potential targets for the beneficial effects of AZA/RES in ameliorating stem cell dysfunctions.     

Identification of nitrogen sources and transformations within karst springs using isotope tracers of nitrogen

Isotope analyses of nitrate and algae were used to gain better understanding of sources of nitrate to Florida’s karst springs and processes affecting nitrate in the Floridan aquifer at multiple scales. In wet years, δ¹⁵N and δ¹⁸O of nitrate ranged from +3 to +9‰ in headwater springs in north Florida, indicating nitrification of soil ammonium as the dominant source. With below normal rainfall, the δ¹⁵N and δ¹⁸O of nitrate were higher in almost all springs (reaching +20.2 and +15.3‰, respectively) and were negatively correlated with dissolved oxygen. In springs with values of δ¹⁵N-NO₃ and δ¹⁸O-NO₃ greater than +10‰, nitrate concentrations declined 40–50% in dry years and variations in the δ¹⁵N and δ¹⁸O of nitrate were consistent with the effects of denitrification. Modeling of the aquifer as a closed system yielded in situ fractionation caused by denitrification of 9 and 18‰ for Δ¹⁸O and Δ¹⁵N, respectively. We observed no strong evidence for local sources of nitrate along spring runs; concentrations declined downstream (0.42–3.3?μmol-NO₃ L^?1 per km) and the isotopic dynamics of algae and nitrate indicated a closed system. Correlation between the δ¹⁵N composition of nitrate and algae was observed at regional and spring-run scales, but the relationship was complicated by varying isotopic fractionation factors associated with nitrate uptake (Δ ranged from 2 to 13‰). Our study demonstrates that nitrate inputs to Florida’s springs are derived predominantly from non-point sources and that denitrification is detectable in aquifer waters with relatively long residence time (i.e., matrix flow).     

The effect of high-fat diet and inhibition of ceramide production on insulin action in liver

Liver, as one of the most important organs involved in lipids and glucose metabolism, is perceived as a key tissue for pharmacotherapy of insulin resistance (IRes) and type 2 diabetes. Ceramides (Cer) are biologically active lipids, which accumulation is associated with the induction of muscle IRes. We sought to determine the role of intrahepatic bioactive lipids production on insulin action in liver of insulin-resistant rats and after myriocin administration. The experiments were conducted on male Wistar rats divided into three groups: Control, fed high-fat diet (HFD), and fed HFD and treated with myriocin (HFD/Myr). Before sacrifice, the animals were infused with a [U-¹³C]palmitate to calculate lipid synthesis rate by means of tracer incorporation technique in particular lipid groups. Liver Cer, diacylglycerols (DAG), acyl-carnitine concentration, and isotopic enrichment were analyzed by LC/MS/MS. Proteins involved in lipid metabolism and insulin pathway were analyzed by western blot analysis. An OGTT and ITT was also performed. HFD-induced IRes and increased both the synthesis rate and the content of DAG and Cer, which was accompanied by inhibition of an insulin pathway. Interestingly, myriocin treatment reduced synthesis rate not only of Cer but also DAG and improved insulin sensitivity. We conclude that the insulin-sensitizing action of myriocin in the liver is a result of the lack of inhibitory effect of lipids on the insulin pathway, due to the reduction of their synthesis rate. This is the first study showing how the synthesis rate of individual lipid groups in liver changes after myriocin administration.