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Borsuk G.,Lublin University of Life Sciences | Strachecka A.,Lublin University of Life Sciences | Olszewski K.,Lublin University of Life Sciences | Paleolog J.,Lublin University of Life Sciences | And 2 more authors.
Biologia (Poland) | Year: 2013

During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3. 0 and 12. 0, and a fall at pH 7. 0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation. © 2013 Versita Warsaw and Springer-Verlag Wien.

Poleszak E.,Medical University of Lublin | Szopa A.,Medical University of Lublin | Wyska E.,Jagiellonian University | Wosko S.,Medical University of Lublin | And 5 more authors.
Life Sciences | Year: 2015

Aims: Worrying data indicate that excessive caffeine intake applies to patients suffering from mental disorders, including depression. It is thus possible to demonstrate the usefulness of caffeine and its derivatives in the treatment of depression. The main goal of the present studywas to evaluate the influence of caffeine (5mg/kg) on the activity of moclobemide (1.5 mg/kg), venlafaxine (1 mg/kg), bupropion (10 mg/kg), and milnacipran (1.25 mg/kg). Moreover, we assessed the influence of caffeine on their serum and brain levels using highperformance liquid chromatography. Main methods: The experiment was carried out on nave adult male Albino Swiss mice. Caffeine and tested drugs were administered intraperitoneally. The influence of caffeine on the activity of selected antidepressant drugs was evaluated in forced swim test (FST). Locomotor activity was estimated to verify and exclude false positive/negative results. To assess the influence of caffeine on the levels of studied antidepressant drugs, their concentrations were determined in murine serum and brains using high-performance liquid chromatography. Key findings: Caffeine potentiated activity of all antidepressants examined in FST and the observed effects were not due to the increase in locomotor activity in the animals. Only in the case of co-administration of caffeine and milnacipran an increased milnacipran concentration in serum was observed without affecting its concentration in the brain. Significance: Caffeine potentiates the activity of antidepressant drugs from different chemical groups. The interactions of caffeine with venlafaxine, bupropion and moclobemide occur in pharmacodynamic phase, whereas the interaction of caffeine-milnacipran occurs, at least partially, in pharmacokinetic phase. © 2015 Elsevier Inc. All rights reserved.

Wojda I.,Institute of Biology and Biochemistry | Taszlow P.,Institute of Biology and Biochemistry
Journal of Insect Physiology | Year: 2013

We report that Galleria mellonella larvae exposed to heat shock was more resistant to infection with entomopathogenic bacteria Bacillus thuringiensis. The insects were exposed to a temperature of 40. °C for 30. min directly before injection of vegetative bacterial cells. It appeared that the kinetics of the immune response was affected in heat-shocked animals. The infection-induced antimicrobial activity of larval hemolymph was stronger in shocked animals in comparison to the non-shocked ones. Hemolymph proteins of molecular weight below 10. kDa, corresponding to the size of antimicrobial peptides, were responsible for this activity. Furthermore, the transcription level of genes encoding antimicrobial peptides: cecropin, gallerimycin, and galiomycin was increased in the fat bodies of insects exposed to heat shock before infection. On the contrary, the heat-shock treatment did not enhance expression of the metalloproteinase inhibitor-IMPI in the infected animals. The difference in the amount of antimicrobial peptides and, consequently, in the defense activity of insect hemolymph, persisted after the action of bacterial metalloproteinases, which are well-known virulence factors. Furthermore, peptides with antimicrobial activity in the hemolymph of infected larvae pre-exposed to heat shock appeared to be more resistant to proteolytic degradation both in vitro and in vivo. Our results point to the mechanism of cross-protection of thermal stress toward innate immune response. © 2013 Elsevier Ltd.

Wojda I.,Institute of Biology and Biochemistry | Koperwas K.,Institute of Biology and Biochemistry | Jakubowicz T.,Institute of Biology and Biochemistry
Acta Biochimica Polonica | Year: 2014

We followed changes in the level of phospho-MAP kinases in the greater wax moth Galleria mellonella after infection with Bacillus thuringiensis. We observed an enhanced level of phosphorylated p38 and JNK in fat bodies of the infected larvae. In hemocytes, injection of B. thuringiensis caused the highest increase in phospho-JNK, however, all pathways were activated after aseptic injection. We report that Galleria mellonella larvae exposed to heat shock before infection showed an enhanced level of phosphorylated JNK in fat body. This finding is relevant in the light of our previous reports, which submit evidence that pre-shocked animals are more resistant to infection.

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