Benzodiazepine Binding to Cultured Human Pituitary Cells

Benzodiazepine receptors were investigated in a cell line of human pituitary cells (18-54,SF) grown in serum-free medium. Preparations of 18-54,SF whole cells and cell membranes were shown to possess saturable [3H]diazepam binding sites. Membrane sites were found to have a KD of 20 nM for diazepam while whole cells possessed a twofold higher value. The KD values determined from Rosenthal, Hill, and kinetic analyses were consistent for each preparation. Whole-cell binding of [3H]diazepam was observed to be more stable than binding to membranes at higher temperatures (37 degrees C) and when longer incubation times (60 min) were employed at 4 degrees C. The rank order potency of various benzodiazepines to inhibit [3H]diazepam binding to whole cells and membranes was Ro 5-4864, flunitrazepam, diazepam, and clonazepam. Representatives of other drug classes did not inhibit this benzodiazepine binding. When 18-54,SF cells were grown for 24 h with 100 nM diazepam and then extensively washed membranes prepared, the KD for diazepam increased to 38 nM whereas the Bmax was unchanged when compared with untreated controls. Overall, these findings indicate that pituitary cells possess a peripheral-type benzodiazepine receptor and that the whole cell receptor differs quantitatively when compared with the membrane receptor.     

Interactions between Sclerostin and Glycosaminoglycans

Sclerostin (SOST) is a glycoprotein having many important functions in the regulation of bone formation as a key negative regulator of Wnt signaling in bone. Surface plasmon resonance (SPR), which allows for a direct quantitative analysis of the label-free molecular interactions in real-time, has been widely used for the biophysical characterization of glycosaminoglycan (GAG)-protein interactions. In the present study, we report kinetics, structural analysis and the effects of physiological conditions (e.g., salt concentrations, Ca2+ and Zn2+concentrations) on the interactions between GAGs and recombinant human (rh) and recombinant mouse (rm) SOST using SPR. SPR results revealed that both SOSTs bind heparin with high affinity (rhSOST-heparin, KD~36 nM and rmSOST-heparin, KD~77 nM) and the shortest oligosaccharide of heparin that effectively competes with full size heparin for SOST binding is octadecasaccharide (18mer). This heparin binding protein also interacts with other highly sulfated GAGs including, disulfated-dermatan sulfate and chondroitin sulfate E. In addition, liquid chromatography-mass spectrometry was used to characterize the structure of sulfated GAGs that bound to SOST.     

Structure of Human Cytomegalovirus UL141 Binding to TRAIL-R2 Reveals Novel,Non-canonical Death Receptor Interactions

The TRAIL (TNF-related apoptosis inducing ligand) death receptors (DRs) of the tumor necrosis factor receptor superfamily (TNFRSF) can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV) expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER) and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways.     

Binding of Extracellular Matrix Molecules by Enterococci

The bacterial surfaces of enterococci are not uniform. This fact is confirmed by several studies and by our results when great differences between individual strains with regard to their cell surface hydrophobicity, binding of eight ECM (extracellular matrix) molecules immobilized on latex beads and four selected ECM molecules in microtiter plates were observed. The strains expressing high binding of ECM molecules (e.g., HJ 18, HJ 23, HJ 24, HJ 26, HJ 28, HJ 36, etc.) were found among Enterococcus faecalis and E. faecium by PAA (particle agglutination assay). On the other hand, weak ECM binders (e.g., HJ 21, HJ 32, HJ 34, HJ 38, HJ 39, HJ 42, HJ 43) were also found. A direct correlation was found between porcine mucin and fetuin binding ability of eight selected strains tested in microtiter plates and by PAA. Moreover, the influence of tunicamycin treatment was different because significant (P < 0.001) blocking effect of tunicamycin was observed with two selected strains (HJ 26 and HJ 36), whereas two strains (HJ 18 and HJ 22) were not significantly affected in their fetuin binding. The treatment of six enterococcal strains with proteolytic enzymes, pronase P, and trypsin, and with sodium metaperiodate also significantly (P < 0.001) decreased their fetuin binding. This suggests that both protein and carbohydrate moieties are involved in the binding of immobilized fetuin. However, the influence of these chemicals on the fetuin binding by individual strains was different. Received: 24 May 2002 / Accepted: 2 August 2002     

Binding of Extracellular Matrix Proteins by Enterococci

Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of ¹²⁵I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity. Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin, human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding of ¹²⁵I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers such as dextran sulfate (M _r 5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine, but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface. Received: 22 April 1996 / Accepted: 29 June 1996     

Quantitative Assessment of Bacterial Adhesion to Eukaryotic Cells of Human Origin

An in vitro system to measure the adhesion of bacteria to human, eukaryotic cells was devised. Adhesion indices for test strains of bacteria could be calculated. Significant differences were then observed between various strains of Escherichia coli from a variety of sources, in their ability to adhere. The possible applications of the test, especially for the routine screening of bacteria for adhesion and for inhibitors of attachment, were considered.     

pH-Dependent Binding of Synthetic β-Amyloid Peptides to Glycosaminoglycans

The seinile plaques found within the cerebral cortex and hippocampus of the Alzheimer disease brain contain β-amyloid peptide (Aβ) fibrils that are associated with a variety of macromolecular species, including dermatan sulfate proteoglycan and heparan sulfate proteoglycan. The latter has been shown recently to bind tightly to both amyloid precursor protein and A/β, and this binding has been attributed largely to the interaction of the core protein of heparan sulfate proteoglycan with Aβ and its precursor. Here we have examined the ability of synthetic Aβ s to bind to and interact with the glycosaminoglycan moieties of proteoglycans. Aβ(1–28) associates with heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate. The interaction of these sulfated polysaccharides with the amyloid peptide results in the formation of large aggregates that are readily sedimented by centrifugation. The ability of both Aβ(1–28) and Aβ(1–40) to bind glycosaminoglycans is pH-dependent, with increasing interaction as the pH values fall below neutrality and very little binding at pH 8.0. The pH profile of heparin-induced aggregation of Aβ(1–28) has a midpoint pH of approximately 6.5, suggesting that one or more histidine residues must be protonated for binding to occur. Analysis of the Aβ sequence reveals a consensus heparin-binding domain at residues 12–17, and this motif contains histidines at positions 13 and 14 that may be involved in the interaction with glycosaminoglycans. This hypothesis is supported by the following observations: (a) Aβ(13–17) binds tightly to a heparin affinity column at pH 4.0, but not at pH 8.0; and (b) an Aβ(13–17) in which histidine residues 13 and 14 have been replaced with serines does not bind to a heparin column at either pH 8.0 or 4.0. Together, the data indicate that Aβ is capable of binding to the glycosaminoglycan chains of proteoglycans, and such an interaction may be relevant to the etiology and pathology of Alzheimer's disease.     

Filoviruses Utilize Glycosaminoglycans for Their Attachment to Target Cells

Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelope glycoprotein (GP), responsible for both receptor binding and fusion of the virus envelope with the host cell membrane, has been demonstrated to interact with multiple molecules in order to enhance entry into host cells. Here we have demonstrated that filoviruses utilize glycosaminoglycans, and more specifically heparan sulfate proteoglycans, for their attachment to host cells. This interaction is mediated by GP and does not require the presence of the mucin domain. Both the degree of sulfation and the structure of the carbohydrate backbone play a role in the interaction with filovirus GPs. This new step of filovirus interaction with host cells can potentially be a new target for antiviral strategies. As such, we were able to inhibit filovirus GP-mediated infection using carrageenan, a broad-spectrum microbicide that mimics heparin, and also using the antiviral dendrimeric peptide SB105-A10, which interacts with heparan sulfate, antagonizing the binding of the virus to cells.     

From Single Cells to Tissues: Interactions between the Matrix and Human Breast Cells in Real Time

Background

Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma - epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.

Results

The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.

Conclusions

Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.     

Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells

Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10–80 fold increase, termed “swarming”), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds.     

Binding of Selected Extracellular Matrix Proteins to Enterococci and Streptococcus bovis of Animal Origin

Thirty-three enterococcal strains and 10 Streptococcus bovis strains were investigated for their protein-binding cell surface components. Seven extracellular matrix (ECM) proteins were immobilized on Difco latex beads to detect these components on the surface of all enterococcal strains and eight non-autoaggregating S. bovis strains by a particle agglutination assay (PAA). Twenty-three selected strains were also examined in microtiter plate assays. According to the absorbance readings (A_570nm), 11 strains were classified as nonadherent (A_570nm < 0.1), 10 strains as weakly adherent (0.1 < A_570nm > 0.3), and 2 strains as strongly adherent (A_570nm > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A_570nm readings of microtiter plate assays. Binding of ¹²⁵I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%–30% and of ¹²⁵I-labeled human vitronectin in the range of 9%–33% to streptococci. The binding of ¹²⁵I-labeled ECM proteins to selected strains was much more effectively inhibited by sulfated carbohydrates than by non-sulfated hyaluronic acid, indicating the importance of the sulfate groups of these inhibitors. An inhibition effect of heparin on bLf binding to four selected strains was higher in comparison with fucoidan in the microtiter plates. Thirty-five out of 44 strains had agglutinated rabbit erythrocytes. However, these strains showed no ability to agglutinate bovine or sheep erythrocytes. Received: 28 April 1999 / Accepted: 26 July 1999     

nAChRs Mediate Human Embryonic Stem Cell-Derived Endothelial Cells: Proliferation,Apoptosis, and Angiogenesis

Background

Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).

Methods and Results

To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31⁺ cells (13.7±2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3±1.4%), CD31 (97.2±2.5%), and VE-cadherin (93.7±2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5×10⁶ hESC-ECs treated for 24 hours with nicotine (10⁻⁸ M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 µg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).

Conclusions

This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways.     

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

The 5′-3′ resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5′-3′ DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.     

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche. The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines. Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions. Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs. Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions. However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly. CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs. Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques. In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system. This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.     

Human Langerhans Cells Control Th Cells via Programmed Death-Ligand 1 in Response to Bacterial Stimuli and Nickel-Induced Contact Allergy

Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4⁺T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6⁺/CCR4⁺ T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6⁺ cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6⁺/CCR4⁺ cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.     

Polarized Secretion of Human Corticosteroid Binding Globulin by MDCK and BeWo Cells

Polarized epithelial cells are able to faithfully direct certain secretory protein components to either their apical or basolateral environments. The mechanism by which these cells accomplish this is still not entirely understood. It is hypothesized that a membrane-associated "sorting receptor" recognizes an intrinsic signal contained within the sorted protein. This interaction directs the secretory protein into the appropriate domain-specific vesicle for transport to either the apical or basolateral face. The nature of this sorting signal and the recognition receptor have not been established. In an effort to understand this phenomenon, a study was undertaken to ascertain whether human corticosteroid binding globulin (hCBG) contains intrinsic signals capable of directing its secretion to a particular side of polarized epithelial cells. The results of these studies have revealed that hCBG is selectively secreted into the apical environment by both MDCK and BeWo cells. Furthermore, this polarized secretion is unaffected by either (1) agents that inhibit N-linked oligosaccharide processing or (2) lysomotrophic drugs, which alter the intravesicular pH. It is concluded that hCBG possesses an intrinsic signal for apical secretion, which can be recognized by two polarized cell types of differing origins. This signal does not appear to be present in the N-linked oligosaccharide moieties of hCBG nor is it affected hy an elevation of the intravesicular pH within the trans-Golgi network. The use of hCBG-transfected MDCK and BeWo cells constitute a useful model system for the investigation of the signals involved in the sorting of secreted proteins.