Changes in Non-Enzymatic Antioxidants in the Blood Following Anaerobic Exercise in Men and Women

Purpose

The aim of this study was to compare changes in total oxidative status (TOS), total antioxidative capacity (TAC) and the concentration of VitA, VitE, VitC, uric acid (UA), reduced (GSH) and oxidized glutathione (GSSG) in blood within 24 hours following anaerobic exercise (AnEx) among men and women.

Methods

10 women and 10 men performed a 20-second bicycle sprint (AnEx). Concentrations of oxidative stress indicators were measured before AnEx and 3, 15 and 30 minutes and 1 hour afterwards. UA, GSH and GSSH were also measured 24 hours after AnEx. Lactate and H⁺ concentrations were measured before and 3 minutes after AnEx.

Results

The increase in lactate and H⁺ concentrations following AnEx was similar in both sexes. Changes in the concentrations of all oxidative stress indicators were significant and did not differ between men and women. In both sexes, TOS, TAC, TOS/TAC and VitA and VitE concentrations were the highest 3 minutes, VitC concentration was the highest 30 minutes, and UA concentration was the highest 1 hour after AnEx. GSH concentration was significantly lower than the initial concentration from 15 minutes to 24 hour after AnEx. GSSG concentration was significantly higher, while the GSH/GSSG ratio was significantly lower than the initial values 1 hour and 24 hour after AnEx.

Conclusions

With similar changes in lactate and H⁺ concentrations, AnEx induces the same changes in TAC, TOS, TOS/TAC and non-enzymatic antioxidants of low molecular weight in men and women. Oxidative stress lasted at least 24 hours after AnEx.     

MicroRNA-181a/b-1 Is Not Required for Innate γδ NKT Effector Cell Development

Thymic development of αβ T lymphocytes into invariant natural killer (NK) T cells depends on their selection via agonistic lipid antigen presented by CD1d. If successful, newly selected NKT cells gain effector functions already in the thymus. Some γδ T cell subsets also acquire effector functions in the thymus. However, it is not clear whether agonistic TCR stimulation is involved in thymic γδ T cell selection and development. Here we combine two genetic models to address this question. MiR-181a/b-1^–/–mice, which show impaired agonistic T cell selection of invariant αβ NKT cells, were crossed to Tcrd-H2BeGFP reporter mice to monitor selection, intra-thymic expansion and differentiation of γδ T cells. We found that miR-181a/b-1-deficiency had no effect on numbers of thymic γδ T cell or on their differentiation towards an IL-17- or IFN-γ-producing effector phenotype. Also, the composition of peripheral lymph node γδ T cells was not affected by miR-181a/b-1-deficiency. Dendritic epidermal γδ T cells were normally present in knock-out animals. However, we observed elevated frequencies and numbers of γδ NKT cells in the liver, possibly because γδ NKT cells can expand and replace missing αβ NKT cells in peripheral niches. In summary, we investigated the role of miR-181a/b-1 for selection, intrathymic development and homeostasis of γδ T cells. We conclude that miR-181a/b-1-dependent modulation of T cell selection is not critically required for innate development of γδ NKT cells or of any other γδ T cell subtypes.     

The activity of selected glycosidases in salivary gland tumors

The monitoring of the patients after salivary gland tumors surgery is an important clinical issue. Still imperfect diagnostic procedures also remain a challenge for searching new sensitive and specific biomarkers of neoplastic processes in salivary glands. The aim of the presented study was an the assessment of the activity of HEX, with its isoforms HEX-A and HEX-B, GLU, GAL, MAN and FUC in salivary gland tumor tissues in comparison to a healthy salivary gland tissues taken during autopsy. A group of 42 patients with benign and malignant salivary gland tumors, aged 25-65 were examined. Fragments of salivary gland tumor tissue, fragments of healthy tissue removed during autopsy, blood serum and saliva were collected from patients with salivary gland tumors and healthy volunteers. In salivary gland tumor tissue the activity of HEX, HEX-A, HEX-B, GAL, FUC was considerably higher than in comparison to healthy salivary gland tissue and ascending trend of activity of GLU, MAN was also noticed. The activity of all lysosomal exoglycosidases in blood serum in patients with salivary gland tumors was considerably higher in comparison to healthy volunteers blood serum. The considerably higher activity of HEX, HEX-A, GLU, GAL, MAN, FUC and descending trend of activity of HEX-B were noticed in saliva of patients with salivary gland tumors in comparison to healthy volunteers. The assessment of HEX in blood serum and saliva of patients with salivary gland tumor can be possibly used in diagnostics and monitoring of salivary glands tumors.     

Differential expression of Snail1 transcription factor and Snail1-related genes in alveolar and embryonal rhabdomyosarcoma subtypes

Rhabdomyosarcoma (RMS) represents the most common sarcoma of soft tissue among children. Two main RMS subtypes are alveolar (ARMS) and embryonal (ERMS). The major goal of this study was to find differentially expressed genes between RMS subtypes that could explain higher metastatic potential in ARMS and would be useful for the differential diagnosis. Using RQ-PCR analysis we compared expression of Snail1 and Snail-related genes among 7 ARMS and 8 ERMS patients' samples obtained from the primary tumors and among 2 alveolar and 2 embryonal cell lines. Our results show that Snail1 is highly expressed both in ARMS patients' samples and the alveolar cell lines. We also found that the expression of E-Cadherin was downregulated and the expression of Matrix Metalloproteinases 2 and 9 (MMP-2 and MMP-9) was upregulated in ARMS. We assume that, as in many tumors, also in RMS Snail1 acts as a regulator for pathways known for their role in cells' metastasis and that Snail1 activity results in increased MMPs and decreased E-Cadherin expression. Our findings may explain higher ARMS aggressiveness. Moreover, we suggest that further studies should be performed to verify if Snail1 can be considered as a potential target for ARMS therapy.     

Species assortment or habitat filtering: a case study of spider communities on lake islands

Competition theory predicts that species of similar ecological niches are less likely to coexist than species with different niches, a process called species assortment. In contrast, the concept of habitat filtering implies that species with similar ecological requirements should co-occur more often than expected by chance. Here we use environmental and ecological data to assess patterns of co-occurrence of regional communities of spiders distributed across two assemblies of lake islands in northern Poland. We found aggregated and random co-occurrences of species of the same genus and a significant tendency of species segregation across genera. We also found that species of the same genus react similarly to important environmental variables. A comparison of ecological traits of species of the local communities with those expected from a random sample from the regional Polish species pool corroborated partly the habitat filtering hypothesis. On the other hand, we did not find evidence for species assortment. Our results also imply that at least some observed species co-occurrences result from niche differentiation.     

Synthetic substrates for measuring activity of autophagy proteases: Autophagins (Atg4)

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A₂ (LC3B-PLA₂), which upon cleavage releases active PLA₂ for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA₂ substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA₂ substrate afforded substantially higher catalytic rates (k_cat/K_m 5.26 × 10⁵ M⁻¹/sec⁻¹) than Ac-GTFG-AFC peptide (0.92 M⁻¹/sec⁻¹), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.Key words: autophagin, fluorogenic assay, tetrapeptide, phospholipase A2, LC3     

Muskrat (Ondatra zibethicus) decline after the expansion of American mink (Neovison vison) in Poland

Field survey data in Central Poland revealed that the proportion of sites inhabited by muskrats decreased from 44% to 7% over one decade. This corresponded to the decline in hunting bags of muskrat over the whole of Poland. The largest hunting harvest of muskrat was recorded in 1987/1988 (66,416 individuals), the smallest in 2007/2008 (4,567 individuals). The decline in hunting bags occurred in all regions analysed; however, it was most rapid in the north and north-east. Before the expansion of mink, which started in northern Poland at the beginning of the 1980s, muskrat densities in particular regions depended on the availability of aquatic habitats. A comparison of hunting bags of muskrat and American mink in years 2002–2008 indicated a significant negative correlation between the numbers of these two species harvested in seven regions of Poland. The negative correlation between numbers of muskrat and mink suggests that mink predation is one of the most important factors in the decline of the muskrat population in Poland.     

Active creatine kinase is present in matrix vesicles isolated from femurs of chicken embryo: Implications for bone mineralization

Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na⁺, K⁺-ATPase, protein kinase C, or alkaline phosphatase to yield inorganic phosphate (P_i), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase 1 producing inorganic pyrophosphate (PP_i), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization.