The Role of IFITM Proteins in Tick-Borne Encephalitis Virus Infection.

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.

Photoactivated Gallium Porphyrin Reduces Staphylococcus aureus Colonization on the Skin and Suppresses Its Ability to Produce Enterotoxin C and TSST-1.

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 (Ga3+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.

The Role of PKGIα and AMPK Signaling Interplay in the Regulation of Albumin Permeability in Cultured Rat Podocytes.

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.

IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins.

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.

Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos-Impact on Infertility Outcome.

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann−Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.

Gallium Mesoporphyrin IX-Mediated Photodestruction: A Pharmacological Trojan Horse Strategy To Eliminate Multidrug-Resistant Staphylococcus aureus.

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 (Ga3+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.

The PKGIα-Rac1 pathway is a novel regulator of insulin-dependent glucose uptake in cultured rat podocytes.

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.

Rab-dependent vesicular traffic affects female gametophyte development in Arabidopsis.

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 ß-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 Rab Geranylgeranyl Transferase Beta Subunit Is Essential for Embryo and Seed Development in Arabidopsis thaliana.

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.

Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System.

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 ß-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.

Implementation of a Non-Thermal Atmospheric Pressure Plasma for Eradication of Plant Pathogens from a Surface of Economically Important Seeds.

Plant pathogenic bacteria cause significant economic losses in the global food production sector. To secure an adequate amount of high-quality nutrition for the growing human population, novel approaches need to be undertaken to combat plant disease-causing agents. As the currently available methods to eliminate bacterial phytopathogens are scarce, we evaluated the effectiveness and mechanism of action of a non-thermal atmospheric pressure plasma (NTAPP). It was ignited from a dielectric barrier discharge (DBD) operation in a plasma pencil, and applied for the first time for eradication of Dickeya and Pectobacterium spp., inoculated either on glass spheres or mung bean seeds. Furthermore, the impact of the DBD exposure on mung bean seeds germination and seedlings growth was estimated. The observed bacterial inactivation rates exceeded 3.07 logs. The two-minute DBD exposure stimulated by 3-4% the germination rate of mung bean seeds and by 13.4% subsequent early growth of the seedlings. On the contrary, a detrimental action of the four-minute DBD subjection on seed germination and early growth of the sprouts was noted shortly after the treatment. However, this effect was no longer observed or reduced to 9.7% after the 96 h incubation period. Due to the application of optical emission spectrometry (OES), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM), we found that the generated reactive oxygen and nitrogen species (RONS), i.e., N2, N2+, NO, OH, NH, and O, probably led to the denaturation and aggregation of DNA, proteins, and ribosomes. Furthermore, the cellular membrane disrupted, leading to an outflow of the cytoplasm from the DBD-exposed cells. This study suggests the potential applicability of NTAPPs as eco-friendly and innovative plant protection methods.

Extracellular ATP modulates podocyte function through P2Y purinergic receptors and pleiotropic effects on AMPK and cAMP/PKA signaling pathways.

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 P2Y1, 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.

Transmembrane regions of bovine herpesvirus 1-encoded UL49.5 and glycoprotein M regulate complex maturation and ER-Golgi trafficking.

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.

Tunneling Nanotubes as a Novel Route of Cell-to-Cell Spread of Herpesviruses.

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.

New Oxidovanadium(IV) Coordination Complex Containing 2-Methylnitrilotriacetate Ligands Induces Cell Cycle Arrest and Autophagy in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Pancreatic cancer is characterized by one of the lowest five-year survival rates. In search for new treatments, some studies explored several metal complexes as potential anticancer drugs. Therefore, we investigated three newly synthesized oxidovanadium(IV) complexes with 2-methylnitrilotriacetate (bcma3-), N-(2-carbamoylethyl)iminodiacetate (ceida3-) and N-(phosphonomethyl)-iminodiacetate (pmida4-) ligands as potential anticancer compounds using pancreatic cancer cell lines. We measured: Cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and lactate dehydrogenase (LDH) assay; antiproliferative activity by bromodeoxyuridine BrdU assay; reactive oxygen species (ROS) generation and cell cycle analysis by flow cytometry; protein level by Western blot and cellular morphology by confocal laser scanning microscopy. The results showed that these oxidovanadium(IV) complexes were cytotoxic on pancreatic cancer cells (PANC-1 and MIA PaCa2), but not on non-tumor human immortalized pancreas duct epithelial cells (hTERT-HPNE) over the concentration range of 10⁻25 µM, following 48 h incubation. Furthermore, molecular mechanisms of cytotoxicity of [4-NH2-2-Me(Q)H][VO(bcma)(H2O)]2H2O (T1) were dependent on antiproliterative activity, increased ROS generation, cell cycle arrest in G2/M phase with simultaneous triggering of the p53/p21 pathway, binucleation, and induction of autophagy. Our study indicates that oxidovanadium(IV) coordination complexes containing 2-methylnitrilotriacetate ligand are good candidates for preclinical development of novel anticancer drugs targeting pancreatic cancer.

The TRPC6-AMPK Pathway is Involved in Insulin-Dependent Cytoskeleton Reorganization and Glucose Uptake in Cultured Rat Podocytes.

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.

Metformin overcomes high glucose-induced insulin resistance of podocytes by pleiotropic effects on SIRT1 and AMPK.

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.

Insulin increases filtration barrier permeability via TRPC6-dependent activation of PKGIα signaling pathways.

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 Ca2+ overload.

Synthesis and antiviral activity of a novel glycosyl sulfoxide against classical swine fever virus.

A novel compound-2″,3″,4″,6″-tetra-O-acetyl-ß-d-galactopyranosyl-(1→4)-2',3',6'-tri-O-acetyl-1-thio-ß-d-glucopyranosyl-(5-nitro-2-pyridyl) sulfoxide-designated GP6 was synthesized and assayed for cytotoxicity and in vitro antiviral properties against classical swine fever virus (CSFV) in this study. We showed that the examined compound effectively arrested CSFV growth in swine kidney cells (SK6) at a 50% inhibitory concentration (IC50) of 5 ± 0.12 µg/ml without significant toxicity for mammalian cells. Moreover, GP6 reduced the viral E2 and E(rns) glycoproteins expression in a dose-dependent manner. We have excluded the possibility that the inhibitor acts at the replication step of virus life cycle as assessed by monitoring of RNA level in cells and culture medium of SK6 cells after single round of infection as a function of GP6 treatment. Using recombinant E(rns) and E2 proteins of classical swine fever virus produced in baculovirus expression system we have demonstrated that GP6 did not influence glycoprotein production and maturation in insect cells. In contrast to mammalian glycosylation pathway, insect cells support only the ER-dependent early steps of this process. Therefore, we concluded that the late steps of glycosylation process are probably the main targets of GP6. Due to the observed antiviral effect accompanied by low cytotoxicity, this inhibitor represents potential candidate for the development of antiviral agents for anti-flavivirus therapy. Further experiments are needed for investigating whether this compound can be used as a safe antiviral agent against other viruses from unrelated groups.

Modulation of acridine mutagen ICR191 intercalation to DNA by methylxanthines--analysis with mathematical models.

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.