Lack of membrane sex steroid receptors for mediating rapid endocrine responses in molluscan nervous systems.

Despite the lack of endogenous synthesis and relevant nuclear receptors, several papers have been published over the decades claiming that the physiology of mollusks is affected by natural and synthetic sex steroids. With scant evidence for the existence of functional steroid nuclear receptors in mollusks, some scientists have speculated that the effects of steroids might be mediated via membrane receptors (i.e. via non-genomic/non-classical actions) - a mechanism that has been well-characterized in vertebrates. However, no study has yet investigated the ligand-binding ability of such receptor candidates in mollusks. The aim of the present study was to further trace the evolution of the endocrine system by investigating the presence of functional membrane sex steroid receptors in a mollusk, the great pond snail (Lymnaea stagnalis). We detected sequences homologous to the known vertebrate membrane sex steroid receptors in the Lymnaea transcriptome and genome data: G protein-coupled estrogen receptor-1 (GPER1); membrane progestin receptors (mPRs); G protein-coupled receptor family C group 6 member A (GPRC6A); and Zrt- and Irt-like protein 9 (ZIP9). Sequence analyses, including conserved domain analysis, phylogenetics, and transmembrane domain prediction, indicated that the mPR and ZIP9 candidates appeared to be homologs, while the GPER1 and GPRC6A candidates seemed to be non-orthologous receptors. All candidates transiently transfected into HEK293MSR cells were found to be localized at the plasma membrane, confirming that they function as membrane receptors. However, the signaling assays revealed that none of the candidates interacted with the main vertebrate steroid ligands. Our findings strongly suggest that functional membrane sex steroid receptors which would be homologous to the vertebrate ones are not present in Lymnaea. Although further experiments are required on other molluscan model species as well, we propose that both classical and non-classical sex steroid signaling for endocrine responses are specific to chordates, confirming that molluscan and vertebrate endocrine systems are fundamentally different.

DNA Reaction Circuits to Establish Designated Biological Functions in Multicellular Community.

In multicellular organisms, individual cells are coordinated through complex communication networks to accomplish various physiological tasks. Aiming to establish new biological functions in the multicellular community, we used DNA as the building block to develop a cascade of nongenetic reaction circuits to establish a dynamic cell-cell communication network. Utilizing membrane-anchored amphiphilic DNA tetrahedra (TDN) as the nanoscaffold, reaction circuits were incorporated into three unrelated cells in order to uniquely regulate their sense-and-response behaviors. As a proof-of-concept, this step enabled these cells to simulate significant biological events involved in T cell-mediated anticancer immunity. Such events included cancer-associated antigen recognition and the presentation of antigen-presenting cells (APCs), APC-facilitated T cell activation and dissociation, and T cell-mediated cancer targeting and killing. By combining the excellent programmability and molecular recognition ability of DNA, our cell-surface reaction circuits hold promise for mimicking and manipulating many biological processes.

Nanoparticle Anisotropy Increases Targeting Interactions on Live-Cell Membranes.

This paper describes how branch lengths of anisotropic nanoparticles can affect interactions between grafted ligands and cell-membrane receptors. Using live-cell, single-particle tracking, we found that DNA aptamer-gold nanostar nanoconstructs with longer branches showed improved binding efficacy to human epidermal growth factor receptor 2 (HER2) on cancer cell membranes. Inhibiting nanoconstruct-HER2 binding promoted nonspecific interactions, which increased the rotational speed of long-branched nanoconstructs but did not affect that of short-branched constructs. Bivariate analysis of the rotational and translational dynamics showed that longer branch lengths increased the ratio of targeting to nontargeting interactions. We also found that longer branches increased the nanoconstruct-cell interaction times before internalization and decreased intracellular trafficking velocities. Differences in binding efficacy revealed by single-particle dynamics can be attributed to the distinct protein corona distributions on short- and long-branched nanoconstructs, as validated by transmission electron microscopy. Minimal protein adsorption at the high positive curvature tips of long-branched nanoconstructs facilitated binding of DNA aptamer ligands to HER2. Our study reveals the significance of nanoparticle branch length in regulating local chemical environment and interactions with live cells at the single-particle level.

Single-cell SERS imaging of dual cell membrane receptors expression influenced by extracellular matrix stiffness.

Membrane receptors perform a diverse range of cellular functions, accounting for more than half of all drug targets. The mechanical microenvironment regulates cell behaviors and phenotype. However, conventional analysis methods of membrane receptors often ignore the effects of the extracellular matrix stiffness, failing to reveal the heterogeneity of cell membrane receptors expression. Herein, we developed an in-situ surface-enhanced Raman scattering (SERS) imaging method to visualize single-cell membrane receptors on substrates with different stiffness. Two SERS substrates, Au@4-mercaptobenzonitrile@Ag@Sgc8c and Au@4-pethynylaniline@Ag@SYL3c, were employed to specifically target protein tyrosine kinase-7 (PTK7) and epithelial cell adhesion molecule (EpCAM), respectively. The polyacrylamide (PA) gels with tunable stiffness (2.5-25 kPa) were constructed to mimic extracellular matrix. The simultaneous SERS imaging of dual membrane receptors on single cancer cells on substrates with different stiffness was achieved. Our findings reveal decreased expression of PTK7 and EpCAM on cells cultured on stiffer substrates and higher migration ability of the cells. The results elucidate the heterogeneity of membrane receptors expression of cells cultured on the substrates with different stiffness. This single-cell analysis method offers an in-situ platform for investigating the impacts of extracellular matrix stiffness on the expression of membrane receptors, providing insights into the role of cell membrane receptors in cancer metastasis.

Pllans-II: Unveiling the Action Mechanism of a Promising Chemotherapeutic Agent Targeting Cervical Cancer Cell Adhesion and Survival Pathways.

Despite advances in chemotherapeutic drugs used against cervical cancer, available chemotherapy treatments adversely affect the patient's quality of life. For this reason, new molecules from natural sources with antitumor potential and few side effects are required. In previous research, Pllans-II, a phospholipase A2 type-Asp49 from Porthidium lansbergii lansbergii snake venom, has shown selective attack against the HeLa and Ca Ski cervical cancer cell lines. This work suggests that the cytotoxic effect generated by Pllans-II on HeLa cells is triggered without affecting the integrity of the cytoplasmic membrane or depolarizing the mitochondrial membranes. The results allow us to establish that cell death in HeLa is related to the junction blockage between α5ß1 integrins and fibronectin of the extracellular matrix. Pllans-II reduces the cells' ability of adhesion and affects survival and proliferation pathways mediated by intracellular communication with the external environment. Our findings confirmed Pllans-II as a potential prototype for developing a selective chemotherapeutic drug against cervical cancer.

Synthesis and characterization of folate-functionalized silica-based materials and application for bioimaging of cancer cells.

Early-stage detection is a vital factor in the later treatment and prognosis of cancer. Enhancing the sensitivity and specificity of the cancer detection pathological and experimental approaches can affect the morbidity and mortality of this disease. A folic acid (FA)-functionalized silica quantum dots (SiQDs)/KCC-NH2@SiO2 nanomaterials were synthesized and characterized as a bioimaging agent of the MCF 7 cancer cells. These nanoparticles showed biocompatible nature with specificity towards folate receptor (FR)-overexpressed MCF 7 cancer cells. Viability findings suggested that the SiQDs/KCC-NH2@SiO2/FA nanomaterials have nontoxic nature towards the cells in the concentration of 200 µg/mL. Fluorescence microscopy images were utilized to estimate the cell internalization of the nanoparticles and further verified by the flow cytometry technique. The differentiation ability of the nanoparticles was also approved by incubation with FR-negative HEK 293 normal cells. The SiQDs/KCC-NH2@SiO2/FA nanoparticle exhibited high stability, bright and high quantum yield fluorescence emission, proposing as a high-quality material for in vivo bioimaging of FR-overexpressed circulating tumoral cancer cells (CTCs).

Binding sites in the epidermal growth factor receptor are responsible for bisphenol S effects on trophoblast cell invasion.

Bisphenol S (BPS) is an endocrine disrupting chemical and the second most abundant bisphenol detected in humans. We have recently demonstrated that in utero exposure to BPS reduces human placenta cell fusion by interfering with epidermal growth factor (EGF)-dependent EGF receptor (EGFR) activation. Our previous work suggests that this occurs via binding of BPS to the extracellular domain of EGFR. However, whether BPS directly binds to EGFR has not been confirmed. We evaluated the binding ability of BPA, BPF and BPS to EGFR to determine whether EGFR binding is a unique attribute of BPS. To test these hypotheses, we first exposed HTR-8/SVneo cells to BPS, BPA, or BPF, with or without EGF. When co-exposed to EGF, BPS, but not BPA nor BPF, reduced EGFR phosphorylation by ∼60%, demonstrating that only BPS can interfere with EGF-dependent EGFR activation. As this indicates that BPS binding to the extracellular domain is responsible for its effect, we performed a computational search for putative binding sites on the EGFR extracellular domain, and performed ligand docking of BPS, BPA, and BPF at these sites. We identified three sites where polar interactions between positively charged residues and the sulfonyl group of BPS could lead binding selectivity over BPA and BPF. To test whether EGFR mutations at the predicted BPS binding sites (Arg255, Lys454, and Arg297) could prevent BPS's interference on EGFR activation, mutations for each EGFR target amino acids (R255A, R297A, and K454A) were introduced. For variants with R297A or K454A mutations, BPS did not affect EGF-mediated EGFR phosphorylation or EGFR-mediated cell invasion, suggesting that these residues are needed for the BPS antagonism effect on EGFR. In conclusion, BPS, but not BPA or BPF, interferes with EGFR-mediated trophoblast cell functions through binding at Arg297 and Lys454 amino acid residues in the extracellular domain of EGFR.

Many or too many progesterone membrane receptors? Clinical implications.

Several receptors for nongenomically initiated actions of progesterone (P4) exist, namely membrane-associated P4 receptors (MAPRs), membrane progestin receptors (mPRs), receptors for neurosteroids [GABAA receptor (GABAAR), NMDA receptor, sigma-1 and -2 receptors (S1R/S2R)], the classical genomic P4 receptor (PGR), and α/ß hydrolase domain-containing protein 2 (ABHD2). Two drugs related to this field have been approved: brexanolone (Zulresso™) for the treatment of postpartum depression, and ganaxolone (Ztalmy™) for the treatment of CDKL5 deficiency disorder. Both are derivatives of P4 and target the GABAAR. Several other indications are in clinical testing. CT1812 (Elayta™) is also being tested for the treatment of Alzheimer's disease (AD) in Phase 2 clinical trials, targeting the P4 receptor membrane component 1 (PGRMC1)/S2R complex. In this Review, we highlight emerging knowledge on the mechanisms of nongenomically initiated actions of P4 and its derivatives.

Progesterone Can Directly Inhibit the Life Activities of Toxoplasma gondii In Vitro through the Progesterone Receptor Membrane Component (PGRMC).

Toxoplasma gondii (T. gondii), as an opportunistic pathogen, has special pathogenic effects on pregnant animals and humans. Progesterone (P4) is a critical hormone that supports pregnancy, and its levels fluctuate naturally during early pregnancy. However, little is known about the association of host P4 levels with the infectivity and pathogenicity of T. gondii. Our study showed that P4 significantly inhibited the invasion and proliferation of tachyzoites, resulting in abnormal cytoskeletal daughter budding and subsequent autophagy in vitro. To investigate the underlying mechanism, we identified a Toxoplasma gondii progesterone membrane receptor protein (TgPGRMC) that was localized to the mitochondrion and closely related to the effect of P4 on tachyzoites. The knockout of the pgrmc gene conferred resistance to P4 inhibitory effects. Our results prove the direct relationship between P4 single factors and T. gondii in vitro and demonstrate that TgPGRMC is an important link between T. gondii and P4, providing a new direction for research on T. gondii infection during pregnancy.

A review of the immunomodulatory activities of polysaccharides isolated from Panax species.

Panax polysaccharides are biopolymers that are isolated and purified from the roots, stems, leaves, flowers, and fruits of Panax L. plants, which have attracted considerable attention because of their immunomodulatory activities. In this paper, the composition and structural characteristics of purified polysaccharides are reviewed. Moreover, the immunomodulatory activities of polysaccharides are described both in vivo and in vitro. In vitro, Panax polysaccharides exert immunomodulatory functions mainly by activating macrophages, dendritic cells, and the complement system. In vivo, Panax polysaccharides can increase the immune organ indices and stimulate lymphocytes. In addition, this paper also discusses the membrane receptors and various signalling pathways of immune cells. Panax polysaccharides have many beneficial therapeutic effects, including enhancing or activating the immune response, and may be helpful in treating cancer, sepsis, osteoporosis, and other conditions. Panax polysaccharides have the potential for use in the development of novel therapeutic agents or adjuvants with beneficial immunomodulatory properties.

Bisphenol S Impairs Invasion and Proliferation of Extravillous Trophoblasts Cells by Interfering with Epidermal Growth Factor Receptor Signaling.

The placenta supports fetal growth and is vulnerable to exogenous chemical exposures. We have previously demonstrated that exposure to the emerging chemical bisphenol S (BPS) can alter placental endocrine function. Mechanistically, we have demonstrated that BPS interferes with epidermal growth factor receptor (EGFR) signaling, reducing placenta cell fusion. Extravillous trophoblasts (EVTs), a placenta cell type that aids with vascular remodeling, require EGF to invade into the maternal endometrium. We hypothesized that BPS would impair EGF-mediated invasion and proliferation in EVTs. Using human EVTs (HTR-8/SVneo cells), we tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional endpoints of EGFR signaling, including EGF endocytosis, cell invasion and proliferation, and endovascular differentiation. We demonstrated that BPS blocked EGF-induced phosphorylation of EGFR by acting as a competitive antagonist to EGFR. Transwell assay and a three-dimensional microfluidic chip invasion assay revealed that BPS exposure can block EGF-mediated cell invasion. BPS also blocked EGF-mediated proliferation and endovascular differentiation. In conclusion, BPS can prevent EGF-mediated EVT proliferation and invasion through EGFR antagonism. Given the role of EGFR in trophoblast proliferation and differentiation during placental development, our findings suggest that maternal exposure to BPS may contribute to placental dysfunction via EGFR-mediated mechanisms.

Genomic variants reducing expression of two endocytic receptors in 46,XY differences of sex development.

Transporter-dependent steroid hormone uptake into target cells was demonstrated in genetically engineered mice and fruit flies. We hypothesized that mutations in such transporters may cause differences in sex development (DSD) in humans. Exome sequencing was performed in 16 genetically unsolved cases of 46,XY DSD selected from an anonymized collection of 708 lines of genital fibroblasts (GF) that were taken from individuals with incomplete virilization. Selection criteria were based on available biochemical characterization of GF compatible with reduced androgen uptake. Two unrelated individuals were identified with mutations in LDL receptor-related protein 2 (LRP2), a gene previously associated with partial sex steroid insensitivity in mice. Like Lrp2-/- mice, affected individuals had non-descended testes. Western blots on GF confirmed reduced LRP2 expression, and endocytosis of sex hormone-binding globulin was reduced. In three unrelated individuals, two with undescended testes, mutations in another endocytic receptor gene, limb development membrane protein 1 like (LMBR1L), were detected. Two of these individuals had mutations affecting the same codon. In a transfected cell model, mutated LMBR1L showed reduced cell surface expression. Our findings suggest that endocytic androgen uptake in complex with sex hormone-binding globulin is relevant in human. LMBR1L may play a similar role in androgen uptake.

Molecular modeling and dynamics studies of the synthetic small molecule agonists with GPR17 and P2Y1 receptor.

The human Guanine Protein coupled membrane Receptor 17 (hGPR17), an orphan receptor that activates uracil nucleotides and cysteinyl leukotrienes is considered as a crucial target for the neurodegenerative diseases. Yet, the detailed molecular interaction of potential synthetic ligands of GPR17 needs to be characterized. Here, we have studied a comparative analysis on the interaction specificity of GPR17-ligands with hGPR17 and human purinergic G protein-coupled receptor (hP2Y1) receptors. Previously, we have simulated the interaction stability of synthetic ligands such as T0510.3657, AC1MLNKK, and MDL29951 with hGPR17 and hP2Y1 receptor in the lipid environment. In the present work, we have comparatively studied the protein-ligand interaction of hGPR17-T0510.3657 and P2Y1-MRS2500. Sequence analysis and structural superimposition of hGPR17 and hP2Y1 receptor revealed the similarities in the structural arrangement with the local backbone root mean square deviation (RMSD) value of 1.16 Å and global backbone RMSD value of 5.30 Å. The comparative receptor-ligand interaction analysis between hGPR17 and hP2Y1 receptor exposed the distinct binding sites in terms of geometrical properties. Further, the molecular docking of T0510.3657 with the hP2Y1 receptor have shown non-specific interaction. The experimental validation also revealed that Gi-coupled activation of GPR17 by specific ligands leads to the adenylyl cyclase inhibition, while there is no inhibition upon hP2Y1 activation. Overall, the above findings suggest that T0510.3657-GPR17 binding specificity could be further explored for the treatment of numerous neuronal diseases. Communicated by Ramaswamy H. Sarma.

New Insights as to Why Progesterone Receptor Modulators, such as Mifepristone, Seem to Be More Effective in Treating Cancers that Are Devoid of the Classical Nuclear Progesterone Receptor.

Mifepristone treatment for advanced cancer has demonstrated considerable improvement in both length and quality of life in patients who no longer have any other treatment options. The target is the progesterone induced blocking factor (PIBF), which helps the tumor to invade the normal tissue and proliferate and suppress cellular immunity. Most of the benefit has been observed in cancers not associated with the classical nuclear progesterone receptor (nPR). There are data showing that the presence of a nPR may be associated with a better prognosis. Membrane PRs seem to be responsible for PIBF secretion. Mifepristone, possibly fails to block another P associated protein that enables the tumor to proliferate, e.g., the progesterone receptor membrane component-1 (PGRMC-1) protein. One hypothesis is that the nPR helps to inhibit tumor production of PGRMC-1 protein. Thus, mifepristone may inhibit tumor spread by suppressing PIBF, but this may be negated by blocking the nPR, allowing PGRMC-1 levels to increase.

Tear Lipocalin and Lipocalin-Interacting Membrane Receptor.

Tear lipocalin is a primate protein that was recognized as a lipocalin from the homology of the primary sequence. The protein is most concentrated in tears and produced by lacrimal glands. Tear lipocalin is also produced in the tongue, pituitary, prostate, and the tracheobronchial tree. Tear lipocalin has been assigned a multitude of functions. The functions of tear lipocalin are inexorably linked to structural characteristics that are often shared by the lipocalin family. These characteristics result in the binding and or transport of a wide range of small hydrophobic molecules. The cavity of tear lipocalin is formed by eight strands (A-H) that are arranged in a ß-barrel and are joined by loops between the ß-strands. Recently, studies of the solution structure of tear lipocalin have unveiled new structural features such as cation-π interactions, which are extant throughout the lipocalin family. Lipocalin has many unique features that affect ligand specificity. These include a capacious and a flexible cavity with mobile and short overhanging loops. Specific features that confer promiscuity for ligand binding in tear lipocalin will be analyzed. The functions of tear lipocalin include the following: antimicrobial activities, scavenger of toxic and tear disruptive compounds, endonuclease activity, and inhibition of cysteine proteases. In addition, tear lipocalin binds and may modulate lipids in the tears. Such actions support roles as an acceptor for phospholipid transfer protein, heteropolymer formation to alter viscosity, and tear surface interactions. The promiscuous lipid-binding properties of tear lipocalin have created opportunities for its use as a drug carrier. Mutant analogs have been created to bind other molecules such as vascular endothelial growth factor for medicinal use. Tear lipocalin has been touted as a useful biomarker for several diseases including breast cancer, chronic obstructive pulmonary disease, diabetic retinopathy, and keratoconus. The functional possibilities of tear lipocalin dramatically expanded when a putative receptor, lipocalin-interacting membrane receptor was identified. However, opposing studies claim that lipocalin-interacting membrane receptor is not specific for lipocalin. A recent study even suggests a different function for the membrane protein. This controversy will be reviewed in light of gene expression data, which suggest that tear lipocalin has a different tissue distribution than the putative receptor. But the data show lipocalin-interacting membrane receptor is expressed on ocular surface epithelium and that a receptor function here would be rational.

Estrogen-dependent KCa1.1 modulation is essential for retaining neuroexcitation of female-specific subpopulation of myelinated Ah-type baroreceptor neurons in rats.

Female-specific subpopulation of myelinated Ah-type baroreceptor neurons (BRNs) in nodose ganglia is the neuroanatomical base of sexual-dimorphic autonomic control of blood pressure regulation, and KCa1.1 is a key player in modulating the neuroexcitation in nodose ganglia. In this study we investigated the exact mechanisms underlying KCa1.1-mediated neuroexcitation of myelinated Ah-type BRNs in the presence or absence of estrogen. BRNs were isolated from adult ovary intact (OVI) or ovariectomized (OVX) female rats, and identified electrophysiologically and fluorescently. Action potential (AP) and potassium currents were recorded using whole-cell recording. Consistently, myelinated Ah-type BRNs displayed a characteristic discharge pattern and significantly reduced excitability after OVX with narrowed AP duration and faster repolarization largely due to an upregulated iberiotoxin (IbTX)-sensitive component; the changes in AP waveform and repetitive discharge of Ah-types from OVX female rats were reversed by G1 (a selective agonist for estrogen membrane receptor GPR30, 100 nM) and/or IbTX (100 nM). In addition, the effect of G1 on repetitive discharge could be completely blocked by G15 (a selective antagonist for estrogen membrane receptor GPR30, 3 µM). These data suggest that estrogen deficiency by removing ovaries upregulates KCa1.1 channel protein in Ah-type BRNs, and subsequently increases AP repolarization and blunts neuroexcitation through estrogen membrane receptor signaling. Intriguingly, this upregulated KCa1.1 predicted electrophysiologically was confirmed by increased mean fluorescent intensity that was abolished by estrogen treatment. These electrophysiological findings combined with immunostaining and pharmacological manipulations reveal the crucial role of KCa1.1 in modulation of neuroexcitation especially in female-specific subpopulation of myelinated Ah-type BRNs and extend our current understanding of sexual dimorphism of neurocontrol of BP regulation.

Anticancer activity of repurposed hemostatic agent desmopressin on AVPR2-expressing human osteosarcoma.

Osteosarcoma is the most prevalent primary bone malignancy. Due to its high aggressiveness, novel treatment strategies are urgently required to improve survival of patients with osteosarcoma, especially those with advanced disease. Desmopressin (dDAVP) is a widely used blood-saving agent that has been repurposed as an adjuvant agent for cancer management due to its antiangiogenic and antimetastatic properties. dDAVP acts as a selective agonist of the vasopressin membrane receptor type 2 (AVPR2) present in the microvascular endothelium and in some cancer cells, including breast, lung, colorectal and neuroendocrine tumor cells. Despite the fact that dDAVP has demonstrated its antitumor efficacy in a wide variety of tumor types, exploration of its potential anti-osteosarcoma activity has, to the best of our knowledge, not yet been conducted. Therefore, the aim of the present study was to evaluate the preclinical antitumor activity of dDAVP in osteosarcoma. Human MG-63 and U-2 OS osteosarcoma cell lines were used to assess in vitro and in vivo therapeutic effects of dDAVP. At low micromolar concentrations, dDAVP reduced AVPR2-expressing MG-63 cell growth in a concentration-dependent manner. In contrast, dDAVP exhibited no direct cytostatic effect on AVPR2-negative U-2 OS cells. As it would be expected for canonical AVPR2-activation, dDAVP raised intracellular cAMP levels in osteosarcoma cells, and coincubation with phosphodiesterase-inhibitor rolipram indicated synergistic antiproliferative activity. Cytostatic effects were associated with increased apoptosis, reduced mitotic index and impairment of osteosarcoma cell chemotaxis, as evaluated by TUNEL-labeling, mitotic body count in DAPI-stained cultures and Transwell migration assays. Intravenous administration of dDAVP (12 µg/kg; three times per week) to athymic mice bearing rapidly growing MG-63 xenografts, was indicated to be capable of reducing tumor progression after a 4-week treatment. No major alterations in animal weight, biochemical or hematological parameters were associated with dDAVP treatment, confirming its good tolerability and safety. Finally, AVPR2 expression was detected by immunohistochemistry in 66% of all evaluated chemotherapy-naive human conventional osteosarcoma biopsies. Taking these findings into account, repurposed agent dDAVP may represent an interesting therapeutic tool for the management of osteosarcoma. Further preclinical exploration of dDAVP activity on orthotopic or metastatic osteosarcoma models are required.

Mifepristone and PGF2α activate phosphatidylinositol hydrolysis in the ovine corpus luteum.

Two experiments were conducted to determine whether mifepristone (RU486) and PGF2α activate the phosphatidylinositol hydrolysis pathway during the midluteal phase of the ovine estrous cycle. In experiment 1, ewes on day 8 of the cycle were given 10 µg RU486 or vehicle into the ovarian artery with removal of the corpus luteum (CL) after 10 min. Blood collected prior to and after treatment was analyzed for progesterone. Aliquots of CL were incubated with 10 µCi of 3H-inositol and in the presence and absence of PGF2α (10 nM) for 15 min. Exposure of CL to RU486 and PGF2α increased phosphatidylinositol hydrolysis (p < 0.05). Serum progesterone was reduced in both control and RU486-treated ewes (p < 0.05) compared to concentrations before treatments. In experiment 2, aliquots of CL collected from ewes on day 8 of the cycle were incubated with 3H-inositol and exposed to RU486 (2 µM) in the presence and absence of PGF2α (1 µM) for 15 min. Treatments stimulated phosphatidylinositol hydrolysis as in Exp 1 (p < 0.05). Progesterone concentrations in incubation medium were increased in response to RU486 and PGF2α (p < 0.05). Collectively, these data suggest that RU486 and PGF2α act to stimulate phosphatidylinositol hydrolysis in the mature ovine CL.

Analysis of Cytomegalovirus Glycoprotein and Cellular Receptor Interactions.

Human cytomegalovirus (HCMV) entry into host cells is a complex process involving interactions between an array of viral glycoproteins with multiple host cell surface receptors. A significant amount of research has been devoted toward identifying these glycoprotein and cellular receptor interactions as the broad cellular tropism of HCMV suggests a highly regulated yet adaptable process that controls viral binding and penetration. However, deciphering the initial binding and cellular receptor activation events by viral glycoproteins remains challenging. The relatively low abundance of receptors and/or interactions with glycoproteins during viral entry, the hydrophobicity of membrane receptors, and the rapid degradation and recycling of activated receptors have complicated the analysis of HCMV entry and the cellular signaling pathways initiated by HCMV engagement to the host membrane. Here, we describe the different methodologies used in our laboratory and others to analyze the interactions between HCMV glycoproteins and host cellular receptors during the entry stage of the viral life cycle.

Reduced Graphene Oxides Modulate the Expression of Cell Receptors and Voltage-Dependent Ion Channel Genes of Glioblastoma Multiforme.

The development of nanotechnology based on graphene and its derivatives has aroused great scientific interest because of their unusual properties. Graphene (GN) and its derivatives, such as reduced graphene oxide (rGO), exhibit antitumor effects on glioblastoma multiforme (GBM) cells in vitro. The antitumor activity of rGO with different contents of oxygen-containing functional groups and GN was compared. Using FTIR (fourier transform infrared) analysis, the content of individual functional groups (GN/exfoliation (ExF), rGO/thermal (Term), rGO/ammonium thiosulphate (ATS), and rGO/ thiourea dioxide (TUD)) was determined. Cell membrane damage, as well as changes in the cell membrane potential, was analyzed. Additionally, the gene expression of voltage-dependent ion channels (clcn3, clcn6, cacna1b, cacna1d, nalcn, kcne4, kcnj10, and kcnb1) and extracellular receptors was determined. A reduction in the potential of the U87 glioma cell membrane was observed after treatment with rGO/ATS and rGO/TUD flakes. Moreover, it was also demonstrated that major changes in the expression of voltage-dependent ion channel genes were observed in clcn3, nalcn, and kcne4 after treatment with rGO/ATS and rGO/TUD flakes. Furthermore, the GN/ExF, rGO/ATS, and rGO/TUD flakes significantly reduced the expression of extracellular receptors (uPar, CD105) in U87 glioblastoma cells. In conclusion, the cytotoxic mechanism of rGO flakes may depend on the presence and types of oxygen-containing functional groups, which are more abundant in rGO compared to GN.