38 results on '"Rychłowski M"'
Search Results
2. Emerin mislocalization during chromatin bridge resolution can drive prostate cancer cell invasiveness in a collagen-rich microenvironment.
- Author
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Popęda M, Kowalski K, Wenta T, Beznoussenko GV, Rychłowski M, Mironov A, Lavagnino Z, Barozzi S, Richert J, Bertolio R, Myszczyński K, Szade J, Bieńkowski M, Miszewski K, Matuszewski M, Żaczek AJ, Braga L, Del Sal G, Bednarz-Knoll N, Maiuri P, and Nastały P
- Abstract
Micronuclei (MN) can form through many mechanisms, including the breakage of aberrant cytokinetic chromatin bridges. The frequent observation of MN in tumors suggests that they might not merely be passive elements but could instead play active roles in tumor progression. Here, we propose a mechanism through which the presence of micronuclei could induce specific phenotypic and functional changes in cells and increase the invasive potential of cancer cells. Through the integration of diverse in vitro imaging and molecular techniques supported by clinical samples from patients with prostate cancer (PCa) defined as high-risk by the D'Amico classification, we demonstrate that the resolution of chromosome bridges can result in the accumulation of Emerin and the formation of Emerin-rich MN. These structures are negative for Lamin A/C and positive for the Lamin-B receptor and Sec61β. MN can act as a protein sinks and result in the pauperization of Emerin from the nuclear envelope. The Emerin mislocalization phenotype is associated with a molecular signature that is correlated with a poor prognosis in PCa patients and is enriched in metastatic samples. Emerin mislocalization corresponds with increases in the migratory and invasive potential of tumor cells, especially in a collagen-rich microenvironment. Our study demonstrates that the mislocalization of Emerin to MN results in increased cell invasiveness, thereby worsening patient prognosis., (© 2024. The Author(s).)
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- 2024
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3. IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins.
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Mikitiuk M, Barczyński J, Bielski P, Arciniega M, Tyrcha U, Hec A, Lipińska AD, Rychłowski M, Holak TA, and Sitar T
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- Glycopeptides metabolism, Membrane Proteins metabolism, Lysosomes metabolism, Peptides chemistry, Antibodies, Monoclonal metabolism
- Abstract
Lysosome-targeting chimeras (LYTACs) have recently been developed to facilitate the lysosomal degradation of specific extracellular and transmembrane molecular targets. However, the LYTAC particles described to date are based on glycopeptide conjugates, which are difficult to prepare and produce on a large scale. Here, we report on the development of pure protein LYTACs based on the non-glycosylated IGF2 peptides, which can be readily produced in virtually any facility capable of monoclonal antibody production. These chimeras utilize the IGF2R/CI-M6PR pathway for lysosomal shuttling and, in our illustrative example, target programmed death ligand 1 (PD-L1), eliciting physiological effects analogous to immune checkpoint blockade. Results from in vitro assays significantly exceed the effects of anti-PD-L1 antibodies alone.
- Published
- 2023
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4. Photoactivated Gallium Porphyrin Reduces Staphylococcus aureus Colonization on the Skin and Suppresses Its Ability to Produce Enterotoxin C and TSST-1.
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Szymczak K, Szewczyk G, Rychłowski M, Sarna T, Zhang L, Grinholc M, and Nakonieczna J
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Staphylococcus aureus is a key pathogen in atopic dermatitis (AD) pathogenicity. Over half of AD patients are carriers of S. aureus . Clinical isolates derived from AD patients produce various staphylococcal enterotoxins, such as staphylococcal enterotoxin C or toxic shock syndrome toxin. The production of these virulence factors is correlated with more severe AD. In this study, we propose cationic heme-mimetic gallium porphyrin (Ga
3+ CHP), a novel gallium metalloporphyrin, as an anti-staphylococcal agent that functions through dual mechanisms: a light-dependent mechanism (antimicrobial photodynamic inactivation, aPDI) and a light-independent mechanism (suppressing iron metabolism). Ga3+ CHP has two additive quaternary ammonium groups that increase its water solubility. Furthermore, Ga3+ CHP is an efficient generator of singlet oxygen and can be recognized by heme-target systems such as Isd, which improves the intracellular accumulation of this compound. Ga3+ CHP activated with green light effectively reduced the survival of clinical S. aureus isolates derived from AD patients (>5 log10 CFU/mL) and affected their enterotoxin gene expression. Additionally, there was a decrease in the biological functionality of studied toxins regarding their superantigenicity. In aPDI conditions, there was no pronounced toxicity in HaCaT keratinocytes with both normal and suppressed filaggrin gene expression, which occurs in ∼50% of AD patients. Additionally, no mutagenic activity was observed. Green light-activated gallium metalloporphyrins may be a promising chemotherapeutic to reduce S. aureus colonization on the skin of AD patients.- Published
- 2023
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5. Proteomic response of A549 lung cancer cell line to protein-polysaccharide complex Venetin-1 isolated from earthworm coelomic fluid.
- Author
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Czaplewska P, Bogucka A, Macur K, Rybicka M, Rychłowski M, and Fiołka MJ
- Abstract
Earthworms' celomic fluid has long attracted scientists' interest due to their toxic properties. It has been shown that the elimination of coelomic fluid cytotoxicity to normal human cells was crucial for the generation of the non-toxic Venetin-1 protein-polysaccharide complex, which exhibits selective activity against Candida albicans cells as well as A549 non-small cell lung cancer cells. To find the molecular mechanisms behind the anti-cancer properties of the preparation, this research investigated the proteome response of A549 cells to the presence of Venetin-1. The sequential window acquisition of all theoretical mass spectra (SWATH-MS) methodology was used for the analysis, which allows for a relative quantitative analysis to be carried out without radiolabelling. The results showed that the formulation did not induce significant proteome responses in normal BEAS-2B cells. In the case of the tumour line, 31 proteins were up regulated, and 18 proteins down regulated. Proteins with increased expression in neoplastic cells are mainly associated with the mitochondrion, membrane transport and the endoplasmic reticulum. In the case of altered proteins, Venetin-1 interferes with proteins that stabilise the structures, i.e., keratin, glycolysis/gluconeogenesis and metabolic processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Czaplewska, Bogucka, Macur, Rybicka, Rychłowski and Fiołka.)
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- 2023
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6. Excess filaggrin in keratinocytes is removed by extracellular vesicles to prevent premature death and this mechanism can be hijacked by Staphylococcus aureus in a TLR2-dependent fashion.
- Author
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Kobiela A, Hovhannisyan L, Jurkowska P, de la Serna JB, Bogucka A, Deptuła M, Paul AA, Panek K, Czechowska E, Rychłowski M, Królicka A, Zieliński J, Gabrielsson S, Pikuła M, Trzeciak M, Ogg GS, and Gutowska-Owsiak D
- Subjects
- Humans, Staphylococcus aureus, Toll-Like Receptor 2 metabolism, Filaggrin Proteins, Mortality, Premature, Keratinocytes metabolism, Extracellular Vesicles metabolism, Staphylococcal Infections
- Abstract
Filaggrin (FLG) protein is indispensable for multiple aspects of the epidermal barrier function but its accumulation in a monomeric filaggrin form may initiate premature keratinocytes death; it is unclear how filaggrin levels are controlled before the formation of storing keratohyalin granules. Here we show that keratinocyte-secreted small extracellular vesicles (sEVs) may contain filaggrin-related cargo providing a route of eliminating excess filaggrin from keratinocytes; blocking of sEV release has cytotoxic effects on those cells. Filaggrin-containing sEVs are found in plasma in both healthy individuals and atopic dermatitis patients. Staphylococcus aureus (S. aureus) enhances packaging and secretion of filaggrin-relevant products within the sEVs for enhanced export via a TLR2-mediated mechanism which is also linked to the ubiquitination process. This filaggrin removal system, preventing premature keratinocyte death and epidermal barrier dysfunction, is exploited by S. aureus which promotes filaggrin elimination from the skin that could help safeguard bacterial growth., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)
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- 2023
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7. The Role of PKGIα and AMPK Signaling Interplay in the Regulation of Albumin Permeability in Cultured Rat Podocytes.
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Rachubik P, Rogacka D, Audzeyenka I, Szrejder M, Topolewska A, Rychłowski M, and Piwkowska A
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- Animals, Rats, Adenosine Monophosphate metabolism, AMP-Activated Protein Kinases metabolism, Permeability, Rats, Wistar, RNA, Small Interfering metabolism, Signal Transduction, Cyclic GMP-Dependent Protein Kinase Type I metabolism, Podocytes metabolism, Albumins metabolism
- Abstract
The permeability of the glomerular filtration barrier (GFB) is mainly regulated by podocytes and their foot processes. Protein kinase G type Iα (PKGIα) and adenosine monophosphate-dependent kinase (AMPK) affect the contractile apparatus of podocytes and influence the permeability of the GFB. Therefore, we studied the interplay between PKGIα and AMPK in cultured rat podocytes. The glomerular permeability to albumin and transmembrane FITC-albumin flux decreased in the presence of AMPK activators and increased in the presence of PKG activators. The knockdown of PKGIα or AMPK with small-interfering RNA (siRNA) revealed a mutual interaction between PKGIα and AMPK and influenced podocyte permeability to albumin. Moreover, PKGIα siRNA activated the AMPK-dependent signaling pathway. AMPKα2 siRNA increased basal levels of phosphorylated myosin phosphate target subunit 1 and decreased the phosphorylation of myosin light chain 2. Podocytes that were treated with AMPK or PKG activators were characterized by the different organization of actin filaments within the cell. Our findings suggest that mutual interactions between PKGIα and AMPKα2 regulate the contractile apparatus and permeability of the podocyte monolayer to albumin. Understanding this newly identified molecular mechanism in podocytes provides further insights into the pathogenesis of glomerular disease and novel therapeutic targets for glomerulopathies.
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- 2023
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8. PTEN-induced kinase 1 deficiency alters albumin permeability and insulin signaling in podocytes.
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Audzeyenka I, Rachubik P, Typiak M, Kulesza T, Kalkowska D, Rogacka D, Rychłowski M, Angielski S, Saleem M, and Piwkowska A
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- Albumins metabolism, Glucose metabolism, Humans, Insulin metabolism, PTEN Phosphohydrolase metabolism, Permeability, Signal Transduction, Diabetic Nephropathies metabolism, Hyperglycemia metabolism, Podocytes metabolism, Protein Kinases genetics, Protein Kinases metabolism
- Abstract
Alterations of insulin signaling in diabetes are associated with podocyte injury, proteinuria, and renal failure. Insulin stimulates glucose transport to cells and regulates other intracellular processes that are linked to cellular bioenergetics, such as autophagy, gluconeogenesis, fatty acid metabolism, and mitochondrial homeostasis. The dysfunction of mitochondrial dynamics, including mitochondrial fusion, fission, and mitophagy, has been observed in high glucose-treated podocytes and renal cells from patients with diabetes. Previous studies showed that prolonged hyperglycemia is associated with the development of insulin resistance in podocytes, and high glucose-treated podocytes exhibit an increase in mitochondrial fission and decrease in markers of mitophagy. In the present study, we found that deficiency of the main mitophagy protein PTEN-induced kinase 1 (PINK1) significantly increased albumin permeability and hampered glucose uptake to podocytes. We suggest that PINK1 inhibition impairs the insulin signaling pathway, in which lower levels of phosphorylated Akt and membrane fractions of the insulin receptor and glucose transporter-4 were observed. Moreover, PINK1-depleted podocytes exhibited lower podocin and nephrin expression, thus identifying a potential mechanism whereby albumin leakage increases under hyperglycemic conditions when mitophagy is inhibited. In conclusion, we found that PINK1 plays an essential role in insulin signaling and the maintenance of proper permeability in podocytes. Therefore, PINK1 may be a potential therapeutic target for the treatment or prevention of diabetic nephropathy., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
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- 2022
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9. Gallium Mesoporphyrin IX-Mediated Photodestruction: A Pharmacological Trojan Horse Strategy To Eliminate Multidrug-Resistant Staphylococcus aureus .
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Michalska K, Rychłowski M, Krupińska M, Szewczyk G, Sarna T, and Nakonieczna J
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- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Heme metabolism, Humans, Mesoporphyrins, Photosensitizing Agents chemistry, Photosensitizing Agents pharmacology, Staphylococcus aureus, Anti-Infective Agents pharmacology, Gallium pharmacology, Methicillin-Resistant Staphylococcus aureus, Staphylococcal Infections
- Abstract
One of the factors determining efficient antimicrobial photodynamic inactivation (aPDI) is the accumulation of a light-activated compound, namely, a photosensitizer (PS). Targeted PS recognition is the approach based on the interaction between the membrane receptor on the bacterial surface and the PS, whereas the compound is efficiently accumulated by the same mechanism as the natural ligand. In this study, we showed that gallium mesoporphyrin IX (Ga
3+ MPIX) provided dual functionality─iron metabolism disruption and PS properties in aPDI. Ga3+ MPIX induced efficient (>5log10 reduction in CFU/mL) bacterial photodestruction with excitation in the area of Q band absorption with relatively low eukaryotic cytotoxicity and phototoxicity. The Ga3+ MPIX is recognized by the same systems as haem by the iron-regulated surface determinant (Isd). However, the impairment in the ATPase of the haem detoxification efflux pump was the most sensitive to the Ga3+ MPIX-mediated aPDI phenotype. This indicates that changes within the metalloporphyrin structure (vinyl vs ethyl groups) did not significantly alter the properties of recognition of the compound but influenced its biophysical properties.- Published
- 2022
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10. Radiosensitization of PC3 Prostate Cancer Cells by 5-Thiocyanato-2'-deoxyuridine.
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Zdrowowicz M, Datta M, Rychłowski M, and Rak J
- Abstract
Purpose: The radiosensitizing properties of uracil analogs modified in the C5 position are very interesting in the context of their effectiveness and safety in radiation therapy. Recently, radiation chemical studies have confirmed that 5-thiocyanato-2'-deoxyuridine (SCNdU) undergoes dissociation induced by an excess electron attachment and established this nucleoside as a potential radiosensitizer. In this paper, we verify the sensitizing properties of SCNdU at the cellular level and prove that it can effectively enhance ionizing radiation-induced cellular death., Methods and Materials: Prostate cancer cells were treated with SCNdU and irradiated with X rays. The cytotoxicity of SCNdU was determined by MTT test. Cell proliferation was assessed using a clonogenic assay. Cell cycle analyses, DNA damage, and cell death analyses were performed by flow cytometry., Results: SCNdU treatment significantly suppressed the proliferation and increased the radiosensitivity of prostate cancer cells. The radiosensitizing effect expressed by the dose enhancement factor is equal to 1.69. Simultaneous exposure of cells to SCNdU and radiation causes an increase in the fraction of the most radiosensitive G2/M phase, enhancement of the histone H2A.X phosphorylation level, and apoptosis induction. Finally, SCNdU turned out to be marginally cytotoxic in the absence of ionizing radiation., Conclusions: Our findings indicate that SCNdU treatment enhances the radiosensitivity of prostate cancer cells in a manner associated with the cell cycle regulation, double strand formation, and a slight induction of apoptosis.
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- 2022
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11. The Role of IFITM Proteins in Tick-Borne Encephalitis Virus Infection.
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Chmielewska AM, Gómez-Herranz M, Gach P, Nekulova M, Bagnucka MA, Lipińska AD, Rychłowski M, Hoffmann W, Król E, Vojtesek B, Sloan RD, Bieńkowska-Szewczyk K, Hupp T, and Ball K
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- Amino Acid Sequence, Cell Line, Cytopathogenic Effect, Viral, Disease Resistance genetics, Disease Resistance immunology, Disease Susceptibility, Encephalitis, Tick-Borne genetics, Encephalitis, Tick-Borne immunology, Gene Expression, Gene Knockdown Techniques, Humans, Membrane Proteins chemistry, Membrane Proteins genetics, Multigene Family, Protein Binding, Protein Interaction Domains and Motifs, Virus Replication, Encephalitis Viruses, Tick-Borne physiology, Encephalitis, Tick-Borne metabolism, Encephalitis, Tick-Borne virology, Host-Pathogen Interactions genetics, Host-Pathogen Interactions immunology, Interferons metabolism, Membrane Proteins metabolism
- Abstract
Tick-borne encephalitis virus (TBEV), of the genus Flavivirus , is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.
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- 2022
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12. Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System.
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Jonca J, Waleron M, Czaplewska P, Bogucka A, Steć A, Dziomba S, Jasiecki J, Rychłowski M, and Waleron K
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- Cell Membrane genetics, Cell Membrane ultrastructure, Extracellular Vesicles ultrastructure, Pectobacterium ultrastructure, Plant Diseases genetics, Plant Diseases microbiology, Protein Translocation Systems ultrastructure, Protein Transport genetics, Virulence genetics, Extracellular Vesicles genetics, Host-Pathogen Interactions genetics, Pectobacterium genetics, Protein Translocation Systems genetics
- Abstract
Bacteria of genus Pectobacterium are Gram-negative rods of the family Pectobacteriaceae . They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium . The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produces large MVs (100-300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that the MVs of β-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in Pectobacterium strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an effective secretion system.
- Published
- 2021
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13. The Rab Geranylgeranyl Transferase Beta Subunit Is Essential for Embryo and Seed Development in Arabidopsis thaliana .
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Rojek J, Tucker MR, Rychłowski M, Nowakowska J, and Gutkowska M
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- Alkyl and Aryl Transferases metabolism, Alkyl and Aryl Transferases physiology, Arabidopsis growth & development, Arabidopsis metabolism, Biological Transport, Gene Expression Regulation, Plant, Indoleacetic Acids metabolism, Mutation, Pollen Tube physiology, Seeds growth & development, Seeds metabolism, Arabidopsis enzymology, Seeds enzymology, Signal Transduction
- Abstract
Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.
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- 2021
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14. Antibacterial Activity of Co(III) Complexes with Diamine Chelate Ligands against a Broad Spectrum of Bacteria with a DNA Interaction Mechanism.
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Turecka K, Chylewska A, Rychłowski M, Zakrzewska J, and Waleron K
- Abstract
Cobalt coordination complexes are very attractive compounds for their therapeutic uses as antiviral, antibacterial, antifungal, antiparasitic, or antitumor agents. Two Co(III) complexes with diamine chelate ligands ([CoCl
2 (dap)2 ]Cl ( 1 ) and [CoCl2 (en)2 ]Cl ( 2 )) (where dap = 1,3-diaminopropane, en = ethylenediamine) were synthesized and characterized by elemental analysis, an ATR technique, and a scan method and sequentially tested against Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration results revealed that anaerobic and microaerophilic bacteria were found to be the most sensitive; the serial passages assay presented insignificant increases in bacterial resistance to both compounds after 20 passages. The synergy assay showed a significant reduction in the MIC values of nalidixic acid when combined with Compounds ( 1 ) or ( 2 ). The assessment of cell damage by the complexes was performed using scanning electron microscopy, transmission electron microscopy, and confocal microscopy, which indicated cell membrane permeability, deformation, and altered cell morphology. DNA interaction studies of the Co(III) complexes with plasmid pBR322 using spectrophotometric titration methods revealed that the interaction between Complex ( 1 ) or ( 2 ) and DNA suggested an electrostatic and intercalative mode of binding, respectively. Furthermore, the DNA cleavage ability of compounds by agarose gel electrophoresis showed nuclease activity for both complexes. The results suggest that the effect of the tested compounds against bacteria can be complex.- Published
- 2021
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15. The PKGIα-Rac1 pathway is a novel regulator of insulin-dependent glucose uptake in cultured rat podocytes.
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Audzeyenka I, Rogacka D, Rachubik P, Typiak M, Rychłowski M, Angielski S, and Piwkowska A
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- Actin Cytoskeleton metabolism, Actin Depolymerizing Factors metabolism, Animals, Cells, Cultured, Cyclic GMP-Dependent Protein Kinase Type I genetics, Female, Podocytes enzymology, Protein Transport, Rats, Wistar, Signal Transduction, p21-Activated Kinases metabolism, rac1 GTP-Binding Protein genetics, Rats, Cyclic GMP-Dependent Protein Kinase Type I metabolism, Glucose metabolism, Glucose Transporter Type 4 metabolism, Hypoglycemic Agents pharmacology, Insulin pharmacology, Podocytes drug effects, rac1 GTP-Binding Protein metabolism
- Abstract
Insulin plays a major role in regulating glucose homeostasis in podocytes. Protein kinase G type Iα (PKGIα) plays an important role in regulating glucose uptake in these cells. Rac1 signaling plays an essential role in the reorganization of the actin cytoskeleton and is also essential for insulin-stimulated glucose transport. The experiments were conducted using primary rat podocytes. We performed western blot analysis, evaluated small GTPases activity assays, measured radioactive glucose uptake, and performed immunofluorescence imaging to analyze the role of PKGIα-Rac1 signaling in regulating podocyte function. We also utilized a small-interfering RNA-mediated approach to determine the role of PKGIα and Rac1 in regulating glucose uptake in podocytes. The present study investigated the influence of the PKGI pathway on the insulin-dependent regulation of activity and cellular localization of small guanosine triphosphatases in podocytes. We found that the PKGIα-dependent activation of Rac1 signaling induced activation of the PAK/cofilin pathway and increased insulin-mediated glucose uptake in podocytes. The downregulation of PKGIα or Rac1 expression abolished this effect. Rac1 silencing prevented actin remodeling and GLUT4 translocation close to the cell membrane. These data provide evidence that PKGIα-dependent activation of the Rac1 signaling pathways is a novel regulator of insulin-mediated glucose uptake in cultured rat podocytes., (© 2020 Wiley Periodicals LLC.)
- Published
- 2021
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16. Identification of a Stable, Non-Canonically Regulated Nrf2 Form in Lung Cancer Cells.
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Mikac S, Rychłowski M, Dziadosz A, Szabelska-Beresewicz A, Fahraeus R, Hupp T, and Sznarkowska A
- Abstract
Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2) transcription factor is recognized for its pro-survival and cell protective role upon exposure to oxidative, chemical, or metabolic stresses. Nrf2 controls a number of cellular processes such as proliferation, differentiation, apoptosis, autophagy, lipid synthesis, and metabolism and glucose metabolism and is a target of activation in chronic diseases like diabetes, neurodegenerative, and inflammatory diseases. The dark side of Nrf2 is revealed when its regulation is imbalanced (e.g., via oncogene activation or mutations) and under such conditions constitutively active Nrf2 promotes cancerogenesis, metastasis, and radio- and chemoresistance. When there is no stress, Nrf2 is instantly degraded via Keap1-Cullin 3 (Cul3) pathway but despite this, cells exhibit a basal activation of Nrf2 target genes. It is yet not clear how Nrf2 maintains the expression of its targets under homeostatic conditions. Here, we found a stable 105 kDa Nrf2 form that is resistant to Keap1-Cul3-mediated degradation and translocates to the nucleus of lung cancer cells. RNA-Seq analysis indicate that it might originate from the exon 2 or exon 3-truncated transcripts. This stable 105 kDa Nrf2 form might help explain the constitutive activity of Nrf2 under normal cellular conditions.
- Published
- 2021
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17. Antimicrobial Photodynamic Inactivation Affects the Antibiotic Susceptibility of Enterococcus spp. Clinical Isolates in Biofilm and Planktonic Cultures.
- Author
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Woźniak A, Kruszewska B, Pierański MK, Rychłowski M, and Grinholc M
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- Biofilms drug effects, Ciprofloxacin pharmacology, Combined Modality Therapy, Daptomycin pharmacology, Dose-Response Relationship, Drug, Drug Resistance, Multiple, Bacterial drug effects, Drug Synergism, Enterococcus faecalis growth & development, Enterococcus faecium growth & development, Gentamicins pharmacology, Microbial Sensitivity Tests, Plankton drug effects, Anti-Bacterial Agents pharmacology, Enterococcus faecalis drug effects, Enterococcus faecium drug effects, Photochemotherapy methods, Photosensitizing Agents pharmacology
- Abstract
Enterococcus faecium and Enterococcus faecalis are opportunistic pathogens that can cause a vast variety of nosocomial infections. Moreover, E. faecium belongs to the group of ESKAPE microbes, which are the main cause of hospital-acquired infections and are especially difficult to treat because of their resistance to many antibiotics. Antimicrobial photodynamic inactivation (aPDI) represents an alternative to overcome multidrug resistance problems. This process requires the simultaneous presence of oxygen, visible light, and photosensitizing compounds. In this work, aPDI was used to resensitize Enterococcus spp. isolates to antibiotics. Antibiotic susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations was combined with synergy testing methods recommended by the American Society for Microbiology. Two clinical isolates, E. faecalis and E. faecium , were treated with a combination of aPDI utilizing rose bengal (RB) or fullerene (FL) derivative as photosensitizers, antimicrobial blue light (aBL), and 10 recommended antibiotics. aPDI appeared to significantly impact the survival rate of both isolates, while aBL had no significant effect. The synergy testing results differed between strains and utilized methods. Synergy was observed for RB aPDI in combination with gentamycin, ciprofloxacin and daptomycin against E. faecalis . For E. faecium, synergy was observed between RB aPDI and gentamycin or ciprofloxacin, while for RB aPDI with vancomycin or daptomycin, antagonism was observed. A combination of FL aPDI gives a synergistic effect against E. faecalis only with imipenem. Postantibiotic effect tests for E. faecium demonstrated that this isolate exposed to aPDI in combination with gentamycin, streptomycin, tigecycline, doxycycline, or daptomycin exhibits delayed growth in comparison to untreated bacteria. The results of synergy testing confirmed the effectiveness of aPDI in resensitization of the bacteria to antibiotics, which presents great potential in the treatment of infections caused by multidrug-resistant strains.
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- 2021
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18. Rab-dependent vesicular traffic affects female gametophyte development in Arabidopsis.
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Rojek J, Tucker MR, Pinto SC, Rychłowski M, Lichocka M, Soukupova H, Nowakowska J, Bohdanowicz J, Surmacz G, and Gutkowska M
- Subjects
- Indoleacetic Acids, Ovule genetics, Pollen Tube, Arabidopsis genetics, Arabidopsis Proteins genetics
- Abstract
Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the β-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
- Published
- 2021
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19. The PKGIα/VASP pathway is involved in insulin- and high glucose-dependent regulation of albumin permeability in cultured rat podocytes.
- Author
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Rachubik P, Szrejder M, Audzeyenka I, Rogacka D, Rychłowski M, Angielski S, and Piwkowska A
- Subjects
- Animals, Cell Adhesion Molecules genetics, Cells, Cultured, Cyclic GMP-Dependent Protein Kinase Type I genetics, Female, Hypoglycemic Agents pharmacology, Microfilament Proteins genetics, Phosphoproteins genetics, Phosphorylation, Podocytes cytology, Podocytes drug effects, Rats, Rats, Wistar, Sweetening Agents pharmacology, Albumins metabolism, Cell Adhesion Molecules metabolism, Cell Membrane Permeability drug effects, Cyclic GMP-Dependent Protein Kinase Type I metabolism, Glucose pharmacology, Insulin pharmacology, Microfilament Proteins metabolism, Phosphoproteins metabolism, Podocytes metabolism
- Abstract
Podocytes, the principal component of the glomerular filtration barrier, regulate glomerular permeability to albumin via their contractile properties. Both insulin- and high glucose (HG)-dependent activation of protein kinase G type Iα (PKGIα) cause reorganization of the actin cytoskeleton and podocyte disruption. Vasodilator-stimulated phosphoprotein (VASP) is a substrate for PKGIα and involved in the regulation of actin cytoskeleton dynamics. We investigated the role of the PKGIα/VASP pathway in the regulation of podocyte permeability to albumin. We evaluated changes in high insulin- and/or HG-induced transepithelial albumin flux in cultured rat podocyte monolayers. Expression of PKGIα and downstream proteins was confirmed by western blot and immunofluorescence. We demonstrate that insulin and HG induce changes in the podocyte contractile apparatus via PKGIα-dependent regulation of the VASP phosphorylation state, increase VASP colocalization with PKGIα, and alter the subcellular localization of these proteins in podocytes. Moreover, VASP was implicated in the insulin- and HG-dependent dynamic remodelling of the actin cytoskeleton and, consequently, increased podocyte permeability to albumin under hyperinsulinaemic and hyperglycaemic conditions. These results indicate that insulin- and HG-dependent regulation of albumin permeability is mediated by the PKGIα/VASP pathway in cultured rat podocytes. This molecular mechanism may explain podocytopathy and albuminuria in diabetes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)
- Published
- 2020
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20. Extracellular ATP modulates podocyte function through P2Y purinergic receptors and pleiotropic effects on AMPK and cAMP/PKA signaling pathways.
- Author
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Szrejder M, Rachubik P, Rogacka D, Audzeyenka I, Rychłowski M, Angielski S, and Piwkowska A
- Subjects
- Animals, Female, Podocytes cytology, Rats, Rats, Wistar, rho GTP-Binding Proteins metabolism, AMP-Activated Protein Kinases metabolism, Adenosine Triphosphate pharmacokinetics, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Podocytes metabolism, Receptors, Purinergic P2 metabolism, Second Messenger Systems drug effects
- Abstract
Podocytes and their foot processes interlinked by slit diaphragms, constitute a continuous outermost layer of the glomerular capillary and seem to be crucial for maintaining the integrity of the glomerular filtration barrier. Purinergic signaling is involved in a wide range of physiological processes in the renal system, including regulating glomerular filtration. We evaluated the role of nucleotide receptors in cultured rat podocytes using non-selective P2 receptor agonists and agonists specific for the P2Y
1 , P2Y2 , and P2Y4 receptors. The results showed that extracellular ATP evokes cAMP-dependent pathways through P2 receptors and influences remodeling of the podocyte cytoskeleton and podocyte permeability to albumin via coupling with RhoA signaling. Our findings highlight the relevance of the P2Y4 receptor in protein kinase A-mediated signal transduction to the actin cytoskeleton. We observed increased cAMP concentration and decreased RhoA activity after treatment with a P2Y4 agonist. Moreover, protein kinase A inhibitors reversed P2Y4 -induced changes in RhoA activity and intracellular F-actin staining. P2Y4 stimulation resulted in enhanced AMPK phosphorylation and reduced reactive oxygen species generation. Our findings identify P2Y-PKA-RhoA signaling as the regulatory mechanism of the podocyte contractile apparatus and glomerular filtration. We describe a protection mechanism for the glomerular barrier linked to reduced oxidative stress and reestablished energy balance., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
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21. Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles.
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Wąchalska M, Rychłowski M, Grabowska K, Kowal K, Narajczyk M, Bieńkowska-Szewczyk K, and Lipińska AD
- Abstract
Extracellular vesicles (EVs) are membranous nanoparticles released by cells as vital mediators of intercellular communication. As such, EVs have become an attractive target for pathogens and cancer cells, which can take control over their cargo composition, as well as their trafficking, shaping the pathogenesis. Despite almost four decades of research on EVs, the number of specific and efficient EV labeling methods is limited, and there is still no universal method for the visualization of their transport in living cells. Lipophilic dyes that non-specifically intercalate into the EVs membranes may diffuse to other membranes, leading to the misinterpretation of the results. Here, we propose a palmitoylated fluorescent mNeonGreen (palmNG) protein as an alternative to chemical dyes for EVs visualization. The Branchiostoma lanceolatum -derived mNeonGreen is a brighter, more stable, and less sensitive to laser-induced bleaching alternative to green fluorescent protein (GFP), which makes it a more potent tag in a variety of fluorescence-based techniques. A palmNG-expressing stable human melanoma cell line was generated using retrovirus gene transfer and cell sorting. This protein partially localizes to cellular membranes, and can be detected inside size-exclusion (SEC)-purified EVs. With the use of flow cytometry and fluorescent confocal microscopy, we performed qualitative and quantitative analyses of palmNG-EVs uptake in recipient human hepatoma cells, in comparison to PKH67-labeled vesicles. Our findings confirm that membrane-embedded mNeonGreen can be successfully applied as a tool in EVs transfer and uptake studies.
- Published
- 2020
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22. Cathepsin C is a novel mediator of podocyte and renal injury induced by hyperglycemia.
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Audzeyenka I, Rachubik P, Rogacka D, Typiak M, Kulesza T, Angielski S, Rychłowski M, Wysocka M, Gruba N, Lesner A, Saleem MA, and Piwkowska A
- Subjects
- Animals, Cathepsin C genetics, Diabetic Nephropathies metabolism, Diabetic Nephropathies pathology, Disease Models, Animal, Female, Gene Knockdown Techniques, Glucose metabolism, Humans, Insulin Resistance, Kidney pathology, Membrane Proteins, Metabolic Syndrome, Obesity, Permeability, RNA, Messenger, Rats, Rats, Zucker, Serum Albumin metabolism, Transcriptome, Cathepsin C metabolism, Hyperglycemia metabolism, Kidney injuries, Kidney metabolism, Podocytes metabolism
- Abstract
A growing body of evidence suggests a role of proteolytic enzymes in the development of diabetic nephropathy. Cathepsin C (CatC) is a well-known regulator of inflammatory responses, but its involvement in podocyte and renal injury remains obscure. We used Zucker rats, a genetic model of metabolic syndrome and insulin resistance, to determine the presence, quantity, and activity of CatC in the urine. In addition to the animal study, we used two cellular models, immortalized human podocytes and primary rat podocytes, to determine mRNA and protein expression levels via RT-PCR, Western blot, and confocal microscopy, and to evaluate CatC activity. The role of CatC was analyzed in CatC-depleted podocytes using siRNA and glycolytic flux parameters were obtained from extracellular acidification rate (ECAR) measurements. In functional analyses, podocyte and glomerular permeability to albumin was determined. We found that podocytes express and secrete CatC, and a hyperglycemic environment increases CatC levels and activity. Both high glucose and non-specific activator of CatC phorbol 12-myristate 13-acetate (PMA) diminished nephrin, cofilin, and GLUT4 levels and induced cytoskeletal rearrangements, increasing albumin permeability in podocytes. These negative effects were completely reversed in CatC-depleted podocytes. Moreover, PMA, but not high glucose, increased glycolytic flux in podocytes. Finally, we demonstrated that CatC expression and activity are increased in the urine of diabetic Zucker rats. We propose a novel mechanism of podocyte injury in diabetes, providing deeper insight into the role of CatC in podocyte biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2020
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23. Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules.
- Author
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Grabowska K, Wąchalska M, Graul M, Rychłowski M, Bieńkowska-Szewczyk K, and Lipińska AD
- Subjects
- Animals, Binding Sites, Cell Fractionation, Cell Line, Cell Membrane metabolism, Flow Cytometry, Gene Expression, Herpesviridae Infections immunology, Histocompatibility Antigens Class II chemistry, Histocompatibility Antigens Class II immunology, Host-Pathogen Interactions, Humans, Mice, Protein Binding, Protein Transport, Viral Envelope Proteins chemistry, Alphaherpesvirinae physiology, Extracellular Vesicles metabolism, Herpesviridae Infections metabolism, Herpesviridae Infections virology, Histocompatibility Antigens Class II metabolism, Viral Envelope Proteins metabolism
- Abstract
Herpesvirus envelope glycoprotein B (gB) is one of the best-documented extracellular vesicle (EVs)-incorporated viral proteins. Regarding the sequence and structure conservation between gB homologs, we asked whether bovine herpesvirus-1 (BoHV-1) and pseudorabies virus (PRV)-encoded gB share the property of herpes simplex-1 (HSV-1) gB to be trafficked to EVs and affect major histocompatibility complex (MHC) class II. Our data highlight some conserved and differential features of the three gBs. We demonstrate that mature, fully processed BoHV-1 and PRV gBs localize to EVs isolated from constructed stable cell lines and EVs-enriched fractions from virus-infected cells. gB also shares the ability to co-localize with CD63 and MHC II in late endosomes. However, we report here a differential effect of the HSV-1, BoHV-1, and PRV glycoprotein on the surface MHC II levels, and MHC II loading to EVs in stable cell lines, which may result from their adverse ability to bind HLA-DR, with PRV gB being the most divergent. BoHV-1 and HSV-1 gB could retard HLA-DR exports to the plasma membrane. Our results confirm that the differential effect of gB on MHC II may require various mechanisms, either dependent on its complex formation or on inducing general alterations to the vesicular transport. EVs from virus-infected cells also contained other viral glycoproteins, like gD or gE, and they were enriched in MHC II. As shown for BoHV-1 gB- or BoHV-1-infected cell-derived vesicles, those EVs could bind anti-virus antibodies in ELISA, which supports the immunoregulatory potential of alphaherpesvirus gB.
- Published
- 2020
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24. Metformin reduces TRPC6 expression through AMPK activation and modulates cytoskeleton dynamics in podocytes under diabetic conditions.
- Author
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Szrejder M, Rachubik P, Rogacka D, Audzeyenka I, Rychłowski M, Kreft E, Angielski S, and Piwkowska A
- Subjects
- Animals, Cytoskeleton metabolism, Diabetic Nephropathies metabolism, Female, GTP Phosphohydrolases metabolism, Glomerular Filtration Barrier drug effects, Glomerular Filtration Barrier metabolism, Glucose metabolism, Male, Podocytes metabolism, Rats, Rats, Wistar, Signal Transduction drug effects, AMP-Activated Protein Kinases metabolism, Cytoskeleton drug effects, Diabetes Mellitus, Type 2 metabolism, Metformin pharmacology, Podocytes drug effects, TRPC Cation Channels metabolism
- Abstract
Podocytes have foot processes that comprise an important cellular layer of the glomerular barrier involved in regulating glomerular permeability. The disturbance of podocyte function plays a central role in the development of proteinuria in diabetic nephropathy. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes, and these channels are involved in disturbing the glomerular filtration barrier in diabetes. Metformin is an anti-diabetic drug widely used for treating patients with type 2 diabetes. Recent studies have suggested that the therapeutic effect of metformin might be mediated by AMPK. The precise function of metformin on cellular function and intracellular signaling in podocytes under diabetic conditions is not fully understood. In this study, we demonstrated that metformin normalized TRPC6 expression via AMPKα1 activation in podocytes exposed to high glucose concentrations. A quantitative analysis showed that metformin increased the colocalization of TRPC6 and AMPKα1 subunits from 42% to 61% in standard glucose (SG) medium and from 29% to 52% in high glucose (HG) medium. AMPK activation was also necessary for maintaining appropriate levels of Rho-family small GTPase activity in HG conditions. Moreover, metformin through AMPK activation remodeled cytoskeleton dynamics, and consequently, reduced filtration barrier permeability in diabetic conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2020
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25. Transmembrane regions of bovine herpesvirus 1-encoded UL49.5 and glycoprotein M regulate complex maturation and ER-Golgi trafficking.
- Author
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Graul M, Kisielnicka E, Rychłowski M, Verweij MC, Tobler K, Ackermann M, Wiertz EJHJ, Bieńkowska-Szewczyk K, and Lipińska AD
- Subjects
- Animals, Cattle, Herpesviridae Infections virology, Herpesvirus 1, Bovine chemistry, Herpesvirus 1, Bovine genetics, Membrane Glycoproteins chemistry, Membrane Glycoproteins genetics, Protein Binding, Protein Domains, Protein Transport, Viral Envelope Proteins chemistry, Viral Envelope Proteins genetics, Cattle Diseases virology, Endoplasmic Reticulum virology, Golgi Apparatus virology, Herpesviridae Infections veterinary, Herpesvirus 1, Bovine metabolism, Membrane Glycoproteins metabolism, Viral Envelope Proteins metabolism
- Abstract
Bovine herpesvirus 1 (BoHV-1)-encoded UL49.5 (a homologue of herpesvirus glycoprotein N) can combine different functions, regulated by complex formation with viral glycoprotein M (gM). We aimed to identify the mechanisms governing the immunomodulatory activity of BoHV-1 UL49.5. In this study, we addressed the impact of gM/UL49.5-specific regions on heterodimer formation, folding and trafficking from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) - events previously found to be responsible for abrogation of the UL49.5-mediated inhibition of the transporter associated with antigen processing (TAP). We first established, using viral mutants, that no other viral protein could efficiently compensate for the chaperone function of UL49.5 within the complex. The cytoplasmic tail of gM, containing putative trafficking signals, was dispensable either for ER retention of gM or for the release of the complex. We constructed cell lines with stable co-expression of BoHV-1 gM with chimeric UL49.5 variants, composed of the BoHV-1 N-terminal domain fused to the transmembrane region (TM) from UL49.5 of varicella-zoster virus or TM and the cytoplasmic tail of influenza virus haemagglutinin. Those membrane-anchored N-terminal domains of UL49.5 were sufficient to form a complex, yet gM/UL49.5 folding and ER-TGN trafficking could be affected by the UL49.5 TM sequence. Finally, we found that leucine substitutions in putative glycine zipper motifs within TM helices of gM resulted in strong reduction of complex formation and decreased ability of gM to interfere with UL49.5-mediated major histocompatibility class I downregulation. These findings highlight the importance of gM/UL49.5 transmembrane domains for the biology of this conserved herpesvirus protein complex.
- Published
- 2019
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26. Tunneling Nanotubes as a Novel Route of Cell-to-Cell Spread of Herpesviruses.
- Author
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Panasiuk M, Rychłowski M, Derewońko N, and Bieńkowska-Szewczyk K
- Subjects
- Animals, Capsid Proteins metabolism, Cattle, Cell Communication physiology, Cell Line, Fluorescent Antibody Technique, Green Fluorescent Proteins genetics, Herpesviridae Infections virology, Intercellular Junctions metabolism, Cell Surface Extensions virology, Herpesviridae Infections transmission, Herpesvirus 1, Bovine physiology, Viral Proteins metabolism
- Abstract
Various types of intercellular connections that are essential for communication between cells are often utilized by pathogens. Recently, a new type of cellular connection, consisting of long, thin, actin-rich membrane extensions named tunneling nanotubes (TNTs), has been shown to play an important role in cell-to-cell spread of HIV and influenza virus. In the present report, we show that TNTs are frequently formed by cells infected by an alphaherpesvirus, bovine herpesvirus 1 (BoHV-1). Viral proteins, such as envelope glycoprotein E (gE), capsid protein VP26, and tegument protein Us3, as well as cellular organelles (mitochondria) were detected by immunofluorescence and live-cell imaging of nanotubes formed by bovine primary fibroblasts and oropharynx cells (KOP cells). Time-lapse confocal studies of live cells infected with fluorescently labeled viruses showed that viral particles were transmitted via TNTs. This transfer also occurred in the presence of neutralizing antibodies, which prevented free entry of BoHV-1. We conclude that TNT formation contributes to successful cell-to-cell spread of BoHV-1 and demonstrate for the first time the participation of membrane nanotubes in intercellular transfer of a herpesvirus in live cells. IMPORTANCE Efficient transmission of viral particles between cells is an important factor in successful infection by herpesviruses. Herpesviruses can spread by the free-entry mode or direct cell-to-cell transfer via cell junctions and long extensions of neuronal cells. In this report, we show for the first time that an alphaherpesvirus can also spread between various types of cells using tunneling nanotubes, intercellular connections that are utilized by HIV and other viruses. Live-cell monitoring revealed that viral transmission occurs between the cells of the same type as well as between epithelial cells and fibroblasts. This newly discovered route of herpesviruses spread may contribute to efficient transmission despite the presence of host immune responses, especially after reactivation from latency that developed after primary infection. Long-range communication provided by TNTs may facilitate the spread of herpesviruses between many tissues and organs of an infected organism., (Copyright © 2018 American Society for Microbiology.)
- Published
- 2018
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27. Inhibition of apoptosis in BHV-1-infected cells depends on Us3 serine/threonine kinase and its enzymatic activity.
- Author
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Brzozowska A, Lipińska AD, Derewońko N, Lesiak D, Rychłowski M, Rąbalski Ł, and Bieńkowska-Szewczyk K
- Subjects
- Animals, Cattle, Cell Line, Herpesvirus 1, Bovine genetics, Mutagenesis, Protein Serine-Threonine Kinases genetics, Viral Proteins genetics, bcl-Associated Death Protein metabolism, Apoptosis drug effects, Herpesvirus 1, Bovine enzymology, Herpesvirus 1, Bovine physiology, Host-Pathogen Interactions, Protein Serine-Threonine Kinases metabolism, Viral Proteins metabolism
- Abstract
Us3 protein is a serine/threonine kinase conserved within the Alphaherpesvirinae subfamily of herpesviruses. The Us3 homologs of herpes simplex virus, pseudorabies virus, and bovine herpesvirus type 5 have been shown to block apoptosis triggered by viral infection or exogenous inducers. To determine whether these characteristics are shared by bovine herpesvirus type 1 Us3, we constructed two viral mutants: BHV-1 Us3 deletion mutant (BHV-1ΔUs3) and a kinase-dead mutant (BHV-1KD). Flow cytometry analysis and TUNEL assay clearly demonstrated, that only BHV-1 wild type virus suppressed infection-induced apoptosis and protected cells from apoptosis triggered by exogenous factors: sorbitol or staurosporine. Us3 of BHV-1 was directly capable of blocking apoptosis without the presence of other viral proteins. The presence of Us3 correlated with phosphorylation of BAD, a pro-apoptotic Bcl-2 family member. Our results clearly indicate that BHV-1 Us3 is necessary for efficient blocking of apoptosis triggered by viral infection and exogenous factors., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2018
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28. Metformin overcomes high glucose-induced insulin resistance of podocytes by pleiotropic effects on SIRT1 and AMPK.
- Author
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Rogacka D, Audzeyenka I, Rychłowski M, Rachubik P, Szrejder M, Angielski S, and Piwkowska A
- Subjects
- AMP-Activated Protein Kinases genetics, AMP-Activated Protein Kinases metabolism, Animals, Cells, Cultured, Cytoprotection genetics, Female, Podocytes metabolism, Rats, Wistar, Signal Transduction drug effects, Signal Transduction genetics, Sirtuin 1 genetics, Sirtuin 1 metabolism, Cytoprotection drug effects, Glucose adverse effects, Insulin Resistance genetics, Metformin pharmacology, Podocytes drug effects
- Abstract
Podocyte insulin sensitivity is critical for glomerular function, and the loss of appropriate insulin signaling leads to alterations and disorders featuring diabetic nephropathy. Energy-sensing pathways, such as AMP-dependent protein kinase (AMPK) and protein deacetylase SIRT1, have been shown to play an important role in insulin resistance. The absence of a stimulating effect of insulin on glucose uptake into podocytes after exposure to hyperglycemic conditions has been demonstrated to be related to a decreased level and activity of SIRT1 protein, leading to reduced AMPK phosphorylation. The present work was undertaken to investigate metformin's ability to restore the insulin responsiveness of podocytes by regulating SIRT1 and AMPK activities. Primary rat podocytes cultured with standard or high glucose concentrations for 5days were transfected with siRNAs targeting SIRT1, AMPKα1, or AMPKα2. SIRT1 activity was measured by a fluorometric method. Insulin-stimulated changes in glucose uptake were used to detect insulin resistance. Podocyte permeability was measured by a transmembrane albumin flux assay to examine podocytes functioning. Our results demonstrated that metformin activated SIRT1 and AMPK, prevented hyperglycemia-induced reduction of SIRT1 protein levels, ameliorated glucose uptake into podocytes, and decreased glomerular filtration barrier permeability. Furthermore, metformin activated AMPK in a SIRT1-independent manner, as the increase in AMPK phosphorylation after metformin treatment was not affected by SIRT1 downregulation. Therefore, the potentiating effect of metformin on insulin-resistant podocytes seemed to be dependent on AMPK, as well as SIRT1 activity, establishing multilateral effects of metformin action., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2018
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29. The TRPC6-AMPK Pathway is Involved in Insulin-Dependent Cytoskeleton Reorganization and Glucose Uptake in Cultured Rat Podocytes.
- Author
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Rachubik P, Szrejder M, Rogacka D, Audzeyenka I, Rychłowski M, Angielski S, and Piwkowska A
- Subjects
- AMP-Activated Protein Kinases antagonists & inhibitors, AMP-Activated Protein Kinases chemistry, Actins metabolism, Animals, Calcium metabolism, Cytoskeleton chemistry, Phosphorylation drug effects, Podocytes cytology, Podocytes drug effects, Podocytes metabolism, Protein Subunits antagonists & inhibitors, Protein Subunits chemistry, Protein Subunits metabolism, RNA Interference, RNA, Small Interfering metabolism, Rats, TRPC6 Cation Channel antagonists & inhibitors, TRPC6 Cation Channel genetics, rac1 GTP-Binding Protein metabolism, rho-Associated Kinases metabolism, AMP-Activated Protein Kinases metabolism, Cytoskeleton metabolism, Glucose metabolism, Insulin pharmacology, Signal Transduction drug effects, TRPC6 Cation Channel metabolism
- Abstract
Background/aims: Podocytes are dynamic polarized cells on the surface of glomerular capillaries that are an essential part of the glomerular filtration barrier. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes and are involved in regulating glomerular filtration. Here we investigated whether the AMPK-TRPC6 pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytes., Methods: Western blot and immunofluorescence analysis confirmed AMPKα and TRPC6 expression, the phosphorylation of proteins associated with actin cytoskeleton reorganization (PAK, rac1, and cofilin), and the expression of insulin signaling proteins (Akt, Insulin receptor). Coimmunoprecipitation and immunofluorescence results demonstrated AMPKα/TRPC6 interaction. To ask whether TRPC6 is involved in the insulin regulation of glucose transport, we measured insulin-dependent (1, 2-3H)-deoxy-D-glucose uptake into podocytes after reducing TRPC6 activity pharmacologically and biochemically (TRPC6 siRNA)., Results: The results suggested a key role for the TRPC6 channel in the mediation of insulin-dependent activation of AMPKα2 and glucose uptake. Moreover, AMPK and TRPC6 activation were required to stimulate the Rac1 signaling pathway., Conclusion: These results suggest a potentially important new mechanism that regulates glucose transport in podocytes and that could be injurious during diabetes., (© 2018 The Author(s). Published by S. Karger AG, Basel.)
- Published
- 2018
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30. Insulin increases filtration barrier permeability via TRPC6-dependent activation of PKGIα signaling pathways.
- Author
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Rogacka D, Audzeyenka I, Rachubik P, Rychłowski M, Kasztan M, Jankowski M, Angielski S, and Piwkowska A
- Subjects
- Animals, Enzyme Activation drug effects, Female, Imidazoles pharmacology, Permeability drug effects, Rats, Rats, Wistar, Cyclic GMP-Dependent Protein Kinase Type I metabolism, Glomerular Filtration Barrier metabolism, Insulin pharmacology, Signal Transduction drug effects, TRPC Cation Channels metabolism
- Abstract
Podocytes are dynamic polarized cells on the surface of glomerular capillaries and an essential component of the glomerular filtration barrier. Insulin increases the activation of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. In addition, accumulating evidence suggests that TRPC6 channels are crucial mediators of podocyte calcium handling and involved in the regulation of glomerular filtration. Therefore, we investigated whether TRPC6 is involved in the regulation of filtration barrier permeability by insulin via the PKGIα-dependent manner. TRPC channel inhibitor SKF96365 abolished insulin-dependent glomerular albumin permeability and transepithelial albumin flux in cultured rat podocytes. Insulin-evoked albumin permeability across podocyte monolayers was also blocked using TRPC6 siRNA. The effect of insulin on albumin permeability was mimicked by treating podocytes with TRPC channel activator (oleolyl-2-acetyl-sn-glycerol, OAG). Insulin or OAG treatment rapidly increased the superoxide generation through activation of NADH oxidase. TRPC inhibitor SKF96365 or siRNA knockdown of TRPC6 attenuated insulin-dependent increase of ROS production. Furthermore, TRPC inhibitor or downregulation of TRPC6 blocked insulin-induced rearrangement of the actin cytoskeleton and attenuated oxidative activation of PKGIα and changes in the phosphorylation of PKG target proteins MYPT1 and MLC. Moreover insulin regulated the PKGIα interaction with TRPC6 in cultured rat podocytes. Taken together, our data suggest a key role of TRPC6 channels in the mediation of insulin-dependent activation of PKGIα signaling pathways. Overall, we have identified a potentially important mechanism that may explain disturbances in filtration barrier permeability in many diseases with increased expression of TRPC6 and chronic Ca
2+ overload., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2017
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31. Consequences of lysine auxotrophy for Candida albicans adherence and biofilm formation.
- Author
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Gabriel I and Rychłowski M
- Subjects
- Animals, Candida albicans pathogenicity, Cell Adhesion genetics, Epithelial Cells metabolism, Gene Expression Regulation, Fungal, Humans, Lysine analogs & derivatives, Lysine biosynthesis, Lysine genetics, Mice, NIH 3T3 Cells, Biofilms growth & development, Candida albicans growth & development, Lysine metabolism
- Abstract
A number of factors are known to be involved in Candida albicans virulence, although biofilm development on the surfaces of indwelling medical devices is considered to promote superficial or systemic disease. Based on previously reported up-regulation of saccharopine and acetyllysine in biofilm cells and activation of the lysine biosynthesis/degradation pathway, we investigated the consequences of Candida albicans lysine auxotrophy on adhesion to host tissues and biofilm formation. Our data indicate that mutant strains lysΔ21/lysΔ22, defective in homocitrate synthase, and lysΔ4, defective in homoaconitase activity (the first two α-aminoadipate pathway enzymes), are able to adhere to mouse embryonic fibroblast cells (cell line NIH/3T3) to the same extent as a control strain SC5314. On the other hand, the auxotrophic mutant strains' development on mouse fibroblast monolayers was significantly reduced up to 5 h post infection. Although invasion into human-derived oral epithelial cells was unaltered, both mutant strains formed a significantly different biofilm architecture and demonstrated diminished viability during long term biofilm propagation.
- Published
- 2017
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32. Corrigendum: Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids.
- Author
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Moskot M, Jakóbkiewicz-Banecka J, Kloska A, Smolińska E, Mozolewski P, Malinowska M, Rychłowski M, Banecki B, Węgrzyn G, and Gabig-Cimińska M
- Published
- 2016
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33. Pentoxifylline as a modulator of anticancer drug doxorubicin. Part II: Reduction of doxorubicin DNA binding and alleviation of its biological effects.
- Author
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Gołuński G, Borowik A, Derewońko N, Kawiak A, Rychłowski M, Woziwodzka A, and Piosik J
- Subjects
- Cell Line, Humans, Microscopy, Confocal, Antibiotics, Antineoplastic pharmacology, DNA metabolism, Doxorubicin pharmacology, Pentoxifylline pharmacology
- Abstract
Anticancer drug doxorubicin is commonly used in cancer treatment. However, drug's severe side effects make toxicity reduction important matter. Another biologically active aromatic compound, pentoxifylline, can sequester aromatic compounds in stacking complexes reducing their bioactivity. This work deals with the problem of alleviating doxorubicin side effects by pentoxifylline. We employed a wide spectrum of prokaryotic and eukaryotic cellular assays. In addition, we used the doxorubicin-pentoxifylline mixed association constant to quantitatively assess pentoxifylline influence on the doxorubicin mutagenic activity. Obtained results indicate strong protective effects of pentoxifylline towards doxorubicin, observed on bacteria and human keratinocytes with no such effects observed on the cancer cells. It may be hypothesized that, considering much shorter half-life of pentoxifylline than doxorubicin, simultaneous administration of doxorubicin and pentoxifylline will lead to gradual release of doxorubicin from complexes with pentoxifylline to reach desired therapeutic concentration. Proposed results shed light on the possible doxorubicin chemotherapy modification and its side effects reduction without the loss of its therapeutic potential., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)
- Published
- 2016
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34. Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids.
- Author
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Moskot M, Jakóbkiewicz-Banecka J, Kloska A, Smolińska E, Mozolewski P, Malinowska M, Rychłowski M, Banecki B, Węgrzyn G, and Gabig-Cimińska M
- Subjects
- Case-Control Studies, Cell Count, Cell Proliferation drug effects, Drug Synergism, Fibroblasts metabolism, Fibroblasts pathology, Gene Expression Profiling, Gene Expression Regulation, Glycosaminoglycans biosynthesis, Glycosaminoglycans genetics, Humans, Lysosomes drug effects, Lysosomes genetics, Microarray Analysis, Molecular Sequence Annotation, Mucopolysaccharidosis II metabolism, Mucopolysaccharidosis II mortality, Primary Cell Culture, Fibroblasts drug effects, Genistein pharmacology, Glycosaminoglycans antagonists & inhibitors, Isoflavones pharmacology, Kaempferols pharmacology, Mucopolysaccharidosis II genetics
- Abstract
Flavonoids were found previously to modulate efficiency of synthesis of glycosaminoglycans (GAGs), compounds which are accumulated in cells of patients suffering from mucopolysaccharidoses (MPSs). The aim of this work was to determine effects of different flavonoids (genistein, kaempferol, daidzein) used alone or in combinations, on expression of genes coding for proteins involved in GAG metabolism. Analyses with DNA microarray, followed by real-time qRT-PCR revealed that genistein, kaempferol and combination of these two compounds induced dose- and time-dependent remarkable alterations in transcript profiles of GAG metabolism genes in cultures of wild-type human dermal fibroblasts (HDFa). Interestingly, effects of the mixture of genistein and kaempferol were stronger than those revealed by any of these compounds used alone. Similarly, the most effective reduction in levels of GAG production, in both HDFa and MPS II cells, was observed in the presence of genistein, keampferol and combination of these compounds. Forty five genes were chosen for further verification not only in HDFa, but also in MPS II fibroblasts by using real-time qRT-PCR. Despite effects on GAG metabolism-related genes, we found that genistein, kaempferol and mixture of these compounds significantly stimulated expression of TFEB. Additionally, a decrease in MTOR transcript level was observed at these conditions.
- Published
- 2015
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35. Modulation of acridine mutagen ICR191 intercalation to DNA by methylxanthines--analysis with mathematical models.
- Author
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Gołuński G, Woziwodzka A, Iermak I, Rychłowski M, and Piosik J
- Subjects
- Aminacrine chemistry, Aminacrine pharmacology, Animals, Caffeine pharmacology, Cattle, Drug Antagonism, Epithelial Cells cytology, Epithelial Cells drug effects, Intercalating Agents pharmacology, Kidney cytology, Kidney drug effects, Kinetics, Models, Chemical, Mutagens pharmacology, Nitrogen Mustard Compounds pharmacology, Pentoxifylline pharmacology, Salmonella typhimurium drug effects, Salmonella typhimurium genetics, Theophylline pharmacology, Thermodynamics, Aminacrine analogs & derivatives, Caffeine chemistry, DNA chemistry, Intercalating Agents chemistry, Mutagens chemistry, Nitrogen Mustard Compounds chemistry, Pentoxifylline chemistry, Theophylline chemistry
- Abstract
Caffeine (CAF) and other methylxanthines (MTX) may interact directly with several aromatic, intercalating ligands through mixed stacking aggregation. Formation of such stacking hetero-complexes may decrease their free form concentration and, in consequence, diminish their biological activity, which is often related to their direct interaction with DNA. In this paper interactions of acridine mutagen (ICR191) with DNA in the presence of three MTX: caffeine (CAF), pentoxifylline (PTX) and theophylline (TH) are investigated. Several mathematical models are used to calculate all association constant values and every component concentration in each analyzed mixture. Model McGhee-von Hippel is used to analyze ligand-DNA interaction, and model Zdunek et al.--to analyze ligand-MTX interactions. Finally, two distinct mathematical models are employed to analyze three-component mixture containing ligand, MTX and DNA molecules. The first model describes possible interactions of ligand with DNA and MTX, and rejects direct MTX interactions with DNA. The second model describes all interactions mentioned above and, additionally, allows MTX to interact directly with DNA. Results obtained using these models are similar. However, correspondence of theoretical results to experimental data is better for the first model than the second one. In this paper possible interactions of ICR191 with eukaryotic cell chromatin are also analyzed, showing that CAF reduces acridine mutagen potential to interact directly with cell chromatin. Additionally, it is demonstrated that MTX inhibit mutagenic activity of ICR191 in a dose-dependent manner. Furthermore, biological activity of ICR191-MTX mixtures corresponds with concentration of free mutagen form calculated using appropriate mathematical model., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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36. Point mutations in BHV-1 Us3 gene abolish its ability to induce cytoskeletal changes in various cell types.
- Author
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Brzozowska A, Rychłowski M, Lipińska AD, and Bieńkowska-Szewczyk K
- Subjects
- Animals, Base Sequence, Cattle, Cell Line, Cell Membrane chemistry, Cell Nucleus chemistry, Cytomegalovirus genetics, Microtubules metabolism, Protein Serine-Threonine Kinases analysis, Cytoskeleton metabolism, Herpesvirus 1, Bovine genetics, Herpesvirus 1, Bovine metabolism, Point Mutation, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism
- Abstract
The Us3 gene is conserved among alphaherpesviruses and codes for a protein kinase, a multifunctional protein involved in many phases of virus infection, like nuclear egress, modulation of apoptosis and modification of the cellular cytoskeleton. Bovine herpesvirus (BHV-1), a member of the Alphaherpesvirinae, contains an open reading frame homologous to Us3 of other herpesviruses, which has been identified as a serine/threonine kinase (Takashima, Y., Tamura, H., Xuan, X., Otsuka, H., 1999. Identification of the Us3 gene product of BHV-1 as a protein kinase and characterization of BHV-1 mutants of the Us3 gene. Virus Res. 59, 23-34). To study the activity of BHV-1 Us3, we have cloned its sequence under control of the human cytomegalovirus (HCMV) promoter/enhancer and introduced it into a recombinant baculovirus (Bac Us3). Confocal microscopy analysis showed profound cytoskeletal modifications in various BHV-1-permissive and non-permissive cells transduced with BacUs3. We observed that Us3 expression changed cellular shape and induced formation of long microtubule-containing cell projections, a phenomenon which had also been observed in cells expressing pseudorabies virus Us3. The intracellular localization of Us3 was mostly nuclear but when the protein accumulated it could be detected in the cytoplasm, cell membranes and projections. Mutated forms of BHV-1 Us3 with point mutations near or within the kinase catalytic domain did not affect cell morphology indicating that kinase activity of BHV-1 Us3 is required for its cytoskeleton remodelling function., (Copyright 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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37. Bovine herpesvirus 1 UL49.5 protein inhibits the transporter associated with antigen processing despite complex formation with glycoprotein M.
- Author
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Lipińska AD, Koppers-Lalic D, Rychłowski M, Admiraal P, Rijsewijk FA, Bieńkowska-Szewczyk K, and Wiertz EJ
- Subjects
- ATP-Binding Cassette Transporters, Animals, Cattle, Cell Line, Endoplasmic Reticulum metabolism, Herpesviridae Infections, Herpesvirus 1, Bovine, Humans, Multiprotein Complexes metabolism, Protein Binding, Protein Transport, trans-Golgi Network metabolism, Histocompatibility Antigens Class I metabolism, Viral Envelope Proteins physiology, Viral Proteins metabolism
- Abstract
Bovine herpesvirus 1 (BHV-1) interferes with peptide translocation by the transporter associated with antigen processing (TAP). Recently, the UL49.5 gene product of BHV-1 was identified as the protein responsible for the observed inhibition of TAP. In BHV-1-infected cells and virions, the UL49.5 protein forms a complex with glycoprotein M (gM). Hence, it was investigated whether UL49.5 can combine the interactions with gM and the TAP complex. In cell lines constitutively expressing both UL49.5 and gM, UL49.5 appears to be required for functional processing of gM. Immunofluorescence-confocal laser scanning microscopy demonstrated that both proteins are interdependent for their redistribution from the endoplasmic reticulum to the trans-Golgi network. Remarkably, expression of cloned gM results in the abrogation of the UL49.5-mediated inhibition of TAP and prevents the degradation of the transporter. However, in BHV-1-infected cells, differences in UL49.5 and gM expression kinetics were seen to create a window of opportunity at the early stages of infection, during which time the UL49.5 protein can act on TAP without gM interference. Moreover, in later periods, non-gM-associated UL49.5 can be detected in addition to the UL49.5/gM complex. Thus, it has been deduced that different functions of UL49.5, editing of gM processing and inhibition of TAP, can be combined during BHV-1 infection.
- Published
- 2006
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38. Quantitative determination of creatine kinase release from herring (Clupea harengus) spermatozoa induced by tributyltin.
- Author
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Grzyb K, Rychłowski M, Biegniewska A, and Skorkowski EF
- Subjects
- Animals, Cell Survival drug effects, Dose-Response Relationship, Drug, Electrophoresis, Cellulose Acetate, Isoenzymes, Male, Microscopy, Confocal, Mitochondria, Muscle drug effects, Mitochondria, Muscle enzymology, Muscle, Skeletal drug effects, Muscle, Skeletal enzymology, Spermatozoa pathology, Creatine Kinase metabolism, Fishes, Spermatozoa drug effects, Spermatozoa enzymology, Trialkyltin Compounds toxicity, Water Pollutants, Chemical toxicity
- Abstract
Creatine kinase (CK, ATP creatine phosphotransferase, EC 2.7.3.2) is an enzyme participating in ATP regeneration, which is the primary source of energy in living organisms. We demonstrated that CK from herring spermatozoa has high activity ( approximately 452 micromol/min/g of fresh semen) and has a different electrophoretic mobility from isoenzymes present in skeletal muscle. In our study, we investigated toxic effect of tributyltin (TBT) on herring spermatozoa using a specific sperm viability kit to observe live and dead sperm cells with a confocal microscope. Treatment of herring spermatozoa with TBT caused a time-dependent decrease of viability: 35% nonviable cells with 5 microM TBT and more than 90% nonviable cells with 10 microM TBT after 6 h exposure. We also monitored CK release from damaged spermatozoa into surrounding medium containing different concentrations of TBT. The higher concentration of TBT was used the more CK release from spermatozoa was observed. We suggest that CK could be a good biomarker of sperm cell membranes degradation in the case when lactate dehydrogenase release from permeabilized cells is not possible for rapid determination of the effect of TBT.
- Published
- 2003
- Full Text
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