Melatonin and its metabolites ameliorate UVR-induced mitochondrial oxidative stress in human MNT-1 melanoma cells

Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine (5-MT), possess a variety of functions, including the scavenging of free radicals and the induction of protective or reparative mechanisms in the cell. Their amphiphilic character allows them to cross cellular membranes and reach subcellular organelles, including the mitochondria. Herein, the action of Mel, 6(OH)Mel, and 5-MT in human MNT-1 melanoma cells against ultraviolet B (UVB) radiation was investigated. The dose of 50 mJ/cm2 caused a significant reduction of cell viability up to 48%, while investigated compounds counteracted this deleterious effect. UVB exposure increased catalase activity and led to a simultaneous Ca++ influx (16%), while tested compounds prevented these disturbances. Additional analysis focused on mitochondrial respiration performed in isolated mitochondria from the liver of BALB/cJ mice where Mel, 6(OH)Mel, and 5-MT significantly enhanced the oxidative phosphorylation at the dose of 10−6 M with lower effects seen at 10−9 or 10−4 M. In conclusion, Mel, 6(OH)Mel and 5-MT protect MNT-1 cells, which express melatonin receptors (MT1 and MT2) against UVB-induced oxidative stress and mitochondrial dysfunction, including the uncoupling of oxidative phosphorylation

Differentiation of mesenchymal stem cells stimulated to autophagy

Autofagia to proces, który ma na celu utrzymanie równowagi pomiędzy syntezą i degradacją białek. Autofagia wykształciła się na drodze ewolucji w celach redukcji okresów głodu. Pozwala ona na pozyskanie substratów metabolicznych poprzez degradację organelli komórkowych i większych fragmentów cytoplazmy. Autofagia zaliczana jest do procesów programowanej śmierci komórki, jednak bardzo często umożliwia komórkom przetrwanie warunków stresowych. Rola autofagii została dobrze poznana w komórkach somatycznych, słabiej natomiast w komórkach macierzystych. Wysoki poziom autofagii występuje konstytutywnie w różnych typach komórek macierzystych. Uważa się zatem, że gra ona kluczową rolę w utrzymaniu unikalnych właściwości komórek macierzystych - ich zdolności do samoodnowy, potencjału różnicującego i pozostawania w okresie uśpienia. Badania wskazują na udział autofagii w różnicowaniu komórek macierzystych. Wykazano bowiem podwyższony poziom aktywnego procesu w początkowych etapach różnicowania komórek a jego zahamowanie skutkowało obniżeniem tempa różnicowania.W celu potwierdzenia hipotezy, iż autofagia jest zaangażowana w proces różnicowania ludzkich, mezenchymalnych komórek macierzystych (hMSC, ang. human Mesenchymal Stem Cells) w kierunku osteoblastów, komórki wyizolowane ze szpiku 64–letniego mężczyzny stymulowano do różnicowania, a następnie oceniano poziom autofagii. Weryfikowano hipotezę o roli środowiska hodowlanego, obecności lub braku surowicy, obecności i braku substancji odżywczych, w procesie autofagii i efektywności stymulacji komórek w kierunku osteogenezy.Poziom autofagii określano za pomocą cytometrii przepływowej, barwiąc komórki oranżem akrydyny, obserwacją komórek w mikroskopie konfokalnym, stosując dansylkadawerynę i LysoTracker oraz metodą Western Blot, oznaczając poziom białek związanych z procesem autofagii: LC3II i p70S6K. Zaawansowanie procesu różnicowania komórek poddanych hodowli w zmiennych warunkach środowiskowych, analizowano w oparciu o aktywność zasadowej fosfatazy.Otrzymane wyniki wskazują na konstytutywną obecność autofagii w izolowanych hMSC 64M oraz w trakcie ich różnicowania w kierunku osteoblastów. Nie wykazano różnic w poziomie autofagii pomiędzy komórkami hodowanymi w warunkach surowiczych i bezsurowiczych, co wskazuje na brak roli surowicy w regulacji autofagii w komórkach hMSC 64M. Umieszczenie komórek w środowisku pozbawionym substancji odżywczych, już po dwóch godzinach zwiększa intensywność procesu autofagii, ale nie ma to wpływu na wzrost tempa różnicowania komórek w kierunku osteoblastów. Istotnie wyższe jest jednak tempo różnicowania hMSC 64M w warunkach surowiczych w stosunku do bezsurowiczych, co wykazano na podstawie aktywności zasadowej fosfatazy. Uzyskane wyniki wskazują, że autofagia jest zaangażowana w proces różnicowania hMSC 64M w kierunku osteoblastów, lecz mechanizm jej inicjacji wydaje się różnić od mechanizmu związanego z brakiem substancji odżywczych.Autophagy is a process that aims to maintain a balance between synthesis and degradation of proteins. It evolved as an adaptation to starvation conditions serving cells nutrients via recycling of some nonessential components. Autophagy is classified as the process of programme cell death, however, it allows cells to survive stress conditions. The role of autophagy is well understood in somatic cells, but little is known about its role in stem cells. Autophagy occurs constitutively in various types of stem cells. Therefore, it is considered as a crucial mechanism in maintaining the unique properties of stem cells – their capacity to self-renewal, pluripotency, and quiescence. Some studies have revealed that autophagy might be involved in the differentiation of stem cells. The machinery of the process was found to significantly increase at the early stage of differentiation. It was also demonstrated that inhibition of autophagy resulted in a reduction of the rate of differentiation.To confirm the hypothesis that autophagy is involved in the process of differentiation of human Mesenchymal Stem Cells (hMSC) toward osteoblast, the cells isolated from the bone marrow of 64 year old man were stimulated to osteoblastogenesis with concomitant measurement of their autophagic activity. Moreover, the role of culture medium composition, the presence or the absence of serum/nutrients, in hMSC 64M differentiation was tested tracing autophagy activity and the rate of osteogenesis.The level of autophagy in the cells was determined by flow cytometry after cell staining with acridine orange, cell examination by meas of the confocal microscope in the presence of dansylcadaverine and LysoTracker fluorescent dyes as well as by Western Blot, measuring the level of autophagy associated proteins: LC3II and p70S6K. Cellular alkaline phosphatase activity was measured as a marker of osteogenesis.The results indicate that autophagy occurs constitutively in hMSC 64M and it is active during cell differentiation into osteoblasts. No differences in the level of autophagy were observed between the cells cultured in serum-containing and serum-free conditions. It indicates that the presence of serum in culture medium has no influence on the autophagy level in hMSC 64M. Cell culture in nutrient-deficient media increased autophagy intensity significantly after two hours of treatment. However, it had no effect on the rate of osteoblastogenesis. The activity of alkaline phosphatase was significantly higher in serum-containing conditions when compared to serum-free conditions what indicates serum-containing conditions more favorable for the progress of cell differentiation. The results demonstrate that autophagy is involved in the differentiation process of hMSC 64M into osteoblasts however, the mechanism of autophagy induction appears to be different from the mechanism triggered by the nutrient deficiency

Relationships between nitric oxide, insulin and BMP-2 signaling pathways during osteogenesis

Kość to tkanka żywa, w której zachodzą ciągłe procesy przebudowy. Zachowanie równowagi pomiędzy tworzeniem się nowej tkanki i jej degradacją jest istotne dla utrzymania struktury i właściwości mechanicznych kości. Komórkami odpowiedzialnymi za budowę tkanki kostnej są osteoblasty, natomiast osteoklasty to komórki uczestniczące w jej resorpcji. Istnieje szereg czynników stymulujących różnicowanie mezenchymalnych komórek macierzystych w kierunku osteoblastów a wśród nich białko morfogenetyczne kości (BMP), tlenek azotu (NO) oraz insulina. BMP to glikoproteina szeroko stosowana w celu wzmożenia osteogenezy u ludzi. Podobny potencjał wykazuje insulina oraz NO. Jednakże BMP, NO oraz insulina aktywują kościotworzenie poprzez odmienne ścieżki sygnalizacyjne, które wydają się krzyżować i wzajemnie regulować. Przypuszczalnie w zależności od drogi sygnalizacji wewnątrzkomórkowej a także typu komórki, bądź stadium jej zróżnicowania kościotworzenie będzie promowane lub hamowane. Celem pracy jest próba odnalezienia miejsc wspólnych dla sygnalizacji związanej z powyższymi czynnikami osteogennymi oraz zaproponowanie mechanizmu wzajemnej regulacji tych szlaków. Punktem wspólnym trzech ścieżek sygnalizacyjnych wydaje się być szlak kinaz MAP a w szczególności kinaza ERK, choć jej aktywność i sposób regulacji (pozytywny czy negatywny) jest bardzo kontrowersyjny.Bone is a living tissue where continuous remodeling processes take place. Maintenance of a balance between formation of a new tissue and its degradation is crucial to preserve appropriate structure and mechanical properties of a bone. Osteoblasts are the group of cells responsible for bone development, whereas osteoclasts are involved in bone tissue resorption. There are several factors that stimulate differentiation of mesenchymal stem cells into osteoblasts. The main is bone morphogenetic protein (BMP), nitric oxide (NO) and insulin. BMP is a glycoprotein widely used to enhance osteogenetic processes in humans but insulin and NO reveals similar potential. However, all those factors activate bone formation via distinct signaling pathways that seems to cross each other and mutually regulate. Presumably, due to different routes of intracellular signaling pathway as well as cell type and its differentiation stage, osteogenesis stimulated with BMP, NO or insulin might be promoted or impaired. The aim of the paper is to search for the places characteristic for all signaling pathways mentioned above and to propose a potential mechanism of their co-action. It seems that MAP kinase pathway, presumable ERK kinase is the connection point of BMP, NO and insulin signaling routes. However, positive or negative regulation of osteogenesis by ERK kinase activity is still controversial

Antimicrobial properties of a peptide derived from the male fertility factor kl2 protein of Drosophila melanogaster

Antimicrobial peptides (AMPs) are important components of innate immunity. Here, we report the antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin-p4 peptide. The antimicrobial activity of multifunctional chemerin is mainly associated with a domain localized in the middle of the chemerin sequence, Val66-Pro85 peptide (chemerin-p4). Using bioinformatic tools, we found homologs of the chemerin-p4 peptide in the proteome of D. melanogaster. One of them is MFF-p1, which is a part of the MFF kl2 protein, encoded by the gene male fertility factor kl2 (kl-2) located on the long arm of the Y chromosome. The second detected peptide (Z-p1) is a part of the Zizimin protein belonging to DOCK family, which is involved in cellular signaling processes. After testing the antimicrobial properties of both peptides, we found that only MFF-p1 possesses these properties. Here, we demonstrate its antimicrobial potential both in vitro and in vivo after infecting D. melanogaster with bacteria. MFF-p1 strongly inhibits the viable counts of E. coli and B. subtilis after 2 h of treatment and disrupts bacterial cells. The expression of kl-2 is regulated by exposure to bacteria and by the circadian clock

Daily Regulation of Phototransduction, Circadian Clock, DNA Repair, and Immune Gene Expression by Heme Oxygenase in the Retina of Drosophila

The daily expression of genes and the changes in gene expression after silencing the heme oxygenase (ho) gene were examined in the retina of Drosophila using microarray and SybrGreen qPCR (quantitative polymerase chain reaction) methods. The HO decrease in the morning upregulated 83 genes and downregulated 57 genes. At night, 80 genes were upregulated and 22 were downregulated. The top 20 genes downregulated after ho silencing in the morning modulate phototransduction, immune responses, autophagy, phagocytosis, apoptosis, the carbon monoxide (CO) response, the oxidative stress/UV response, and translation. In turn, the genes that upregulated at night were involved in translation—the response to oxidative stress, DNA damage, and phototransduction. Among the top 20 genes downregulated at night were genes involved in phototransduction, immune responses, and autophagy. For some genes, a low level of HO had an opposite effect in the morning compared to those at night. Silencing ho also changed the expression of circadian clock genes, while the HO decrease during the night enhanced the expression of immune system genes. The results showed that the cyclic expression of HO is important for controlling several processes in the retina, including neuroprotection and those involved in the innate immune system

Secretory leukocyte protease inhibitor is present in circulating and tissue-recruited human eosinophils and regulates their migratory function

Eosinophils and secretory leukocyte protease inhibitor (SLPI) are both associated with Th2 immune responses and allergic diseases, but whether the fact that they are both implicated in these conditions is pathophysiologically related remains unknown. Here we demonstrate that human eosinophils derived from normal individuals are one of the major sources of SLPI among circulating leukocytes. SLPI was found to be stored in the crystalline core of eosinophil granules, and its dislocation/rearrangement in the crystalline core likely resulted in changes in immunostaining for SLPI in these cells. High levels of SLPI were also detected in blood eosinophils from patients with allergy-associated diseases marked by eosinophilia. These include individuals with eosinophilic granulomatosis with polyangiitis (EGPA) and atopic dermatitis (AD), who were also found to have elevated SLPI levels in their plasma. In addition to the circulating eosinophils, diseased skin of AD patients also contained SLPI-positive eosinophils. Exogenous, recombinant SLPI increased numbers of migratory eosinophils and supported their chemotactic response to CCL11, one of the key chemokines that regulate eosinophil migratory cues. Together, these findings suggest a role for SLPI in controlling Th2 pathophysiologic processes via its impact on and/or from eosinophils