B Cell Activation by Outer Membrane Vesicles—A Novel Virulence Mechanism

Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca²⁺ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19⁺ IgD⁺ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.     

Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content.     

Spatiotemporal Detection and Analysis of Exocytosis Reveal Fusion “Hotspots” Organized by the Cytoskeleton in Endocrine Cells

Total internal reflection fluorescence microscope has often been used to study the molecular mechanisms underlying vesicle exocytosis. However, the spatial occurrence of the fusion events within a single cell is not frequently explored due to the lack of sensitive and accurate computer-assisted programs to analyze large image data sets. Here, we have developed an image analysis platform for the nonbiased identification of different types of vesicle fusion events with high accuracy in different cell types. By performing spatiotemporal analysis of stimulus-evoked exocytosis in insulin-secreting INS-1 cells, we statistically prove that individual vesicle fusion events are clustered at hotspots. This spatial pattern disappears upon the disruption of either the actin or the microtubule network; this disruption also severely inhibits evoked exocytosis. By demonstrating that newcomer vesicles are delivered from the cell interior to the surface membrane for exocytosis, we highlight a previously unappreciated mechanism in which the cytoskeleton-dependent transportation of secretory vesicles organizes exocytosis hotspots in endocrine cells.     

Expression and Secretion of TNF-α in Mouse Taste Buds: A Novel Function of a Specific Subset of Type II Taste Cells

Taste buds are chemosensory structures widely distributed on the surface of the oral cavity and larynx. Taste cells, exposed to the oral environment, face great challenges in defense against potential pathogens. While immune cells, such as T-cells and macrophages, are rarely found in taste buds, high levels of expression of some immune-response-associated molecules are observed in taste buds. Yet, the cellular origins of these immune molecules such as cytokines in taste buds remain to be determined. Here, we show that a specific subset of taste cells selectively expresses high levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α). Based on immuno-colocalization experiments using taste-cell-type markers, the TNF-α-producing cells are predominantly type II taste cells expressing the taste receptor T1R3. These cells can rapidly increase TNF-α production and secretion upon inflammatory challenges, both in vivo and in vitro. The lipopolysaccharide (LPS)-induced TNF-α expression in taste cells was completely eliminated in TLR2(-/-)/TLR4(-/-) double-gene-knockout mice, which confirms that the induction of TNF-α in taste buds by LPS is mediated through TLR signaling pathways. The taste-cell-produced TNF-α may contribute to local immune surveillance, as well as regulate taste sensation under normal and pathological conditions.     

A Novel,Volume-correlated Cl− Conductance in Marginal Cells Dissociated from the Stria Vascularis of Gerbils

Using the whole-cell patch-clamp technique, we examined Cl⁻-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl⁻-selective conductance of ∼18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mm Cl⁻. Under a quasi-symmetrical Cl⁻ condition, the `instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic currents was as SCN⁻ > Br⁻≅ Cl⁻ > F⁻ > NO⁻ ₃≅ I⁻ > gluconate⁻, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited volume changes that were accounted for by the Cl⁻ currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular Cl⁻ concentration, anion substitution and altered holding potentials. The Cl⁻ conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl⁻ current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mm) and diphenylamine-2-carboxylic acid (DPC, 1.0 mm), but 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS, 0.5 mm) was without effect. The properties of the present whole-cell Cl⁻ current resembled those of the single Cl⁻ channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res. 83:89–100, 1995), suggesting that the volume-correlated Cl⁻ conductance could be ascribed predominantly to the basolateral membrane. This Cl⁻ conductance may function not only in cell volume regulation but also for the transport of Cl⁻ and the setting of membrane potential in marginal cells under physiological conditions. Received: 15 August 1995/Revised: 3 November 1995     

Synthesis and Anticancer Activity of Novel Derivatives of α,β-Unsaturated Ketones Based on Oleanolic Acid: in Vitro and in Silico Studies against Prostate Cancer Cells

Herein, new derivatives of α,β-unsaturated ketones based on oleanolic acid ( 4 a – i ) were designed, synthesized, characterized, and tested against human prostate cancer (PC3). According to the in vitro cytotoxic study, title compounds ( 4 a – i ) showed significantly lower toxicity toward healthy cells (HUVEC) in comparison with the reference drug doxorubicin. The compounds with the lowest IC₅₀ values on PC3 cell lines were 4 b (7.785 μM), 4 c (8.869 μM), and 4 e (8.765 μM). The results of the ADME calculations showed that the drug-likeness parameters were within the defined ranges according to Lipinski's and Jorgensen's rules. For the most potent compounds 4 b , 4 c , and 4 e , a molecular docking analysis using the induced fit docking (IFD) protocol was performed against three protein targets (PARP, PI3K, and mTOR). Based on the IFD scores, compound 4 b had the highest calculated affinity for PARP1, while compound 4 c had higher affinities for mTOR and PI3K. The MM-GBSA calculations showed that the most potent compounds had high binding affinities and formed stable complexes with the protein targets. Finally, a 50 ns molecular dynamics simulation was performed to study the behavior of protein target complexes under in silico physiological conditions.     

Effects on Interactions of Oppositely Charged Phospholipid Vesicles of Covalent Attachment of Polyethylene Glycol Oligomers to Their Surfaces: Adhesion, Hemifusion, Full Fusion and “Endocytosis”

Oppositely charged giant vesicles are known to adhere, hemifuse and fuse, all of which depend upon the nature of surface contacts. To further understand such interactions, vesicles were surface-modified with polyethylene glycol (PEG), a moiety that reduces surface-surface interactions. Positively charged vesicles were composed of O-ethyldioleoylphosphocholine (EDOPC), dioleoylphosphatidylcholine (DOPC) and a carbocyanine dye (DiO), with and without DPPE-PEG (dipalmitoylphosphatidylethanolamine-N-PEG MW of the PEG portion = 2000). Negatively charged vesicles were composed of dioleoylphosphatidylglycerol (DOPG), DOPC and a rhodamine B dye (Rh-PE), with as well as without DPPE-PEG (MW 2,000). A microscope-mounted electrophoresis chamber allowed selected pairs of vesicles to be brought into contact while color images were collected at video rates (30 frames/s). Data collection focused on effects of PEG on vesicle interactions as a function of the surface charge density. Relative to PEG-free preparations, vesicles containing DPPE-PEG (1) formed larger contact zones, (2) underwent adhesion and fusion processes more slowly (by two to four times) and (3) at high charge density were less susceptible to rupture upon contact. Unexpectedly, PEG-containing vesicles exhibited engulfment of a smaller by a larger vesicle, a process topologically similar to cellular endocytosis. These observations are interpreted to mean that, although initial surface-surface interactions are weakened by the intervening layer of PEG chains, eventual and strong bilayer-bilayer contact is still possible, evidently because the lipid anchors of these chains can diffuse away from the contact zone.     

Elevated Membrane and Soluble CD64: A Novel Marker Reflecting Altered FcγR Function and Disease in Early Rheumatoid Arthritis That Can Be Regulated by Anti-Rheumatic Treatment

Objectives

Fc receptors (FcR) interacting with immune complexes (ICs) is a central event in the immune pathogenesis of rheumatoid arthritis (RA). Here we asked if a specific FcR is linked to RA pathogenesis and if FcR activities relate to disease and treatment outcome in early RA.

Material and Methods

Twenty autoantibody-positive RA patients and 33 HC were included. The patients were evaluated before and after treatment with methotrexate and prednisolone. At follow-up, the EULAR response criteria were applied to determine the individual treatment outcomes. Serum immunoglobulin levels were measured and the expression of FcR for IgG (FcγR) and IgA (FcαR) on peripheral blood monocytes were determined by flow cytometry. The monocytic FcγR function was evaluated by human IgG1 and IgG3 IC-binding and TNFα stimulated release. Plasma levels of soluble FcRs (sFcRs) were determined with ELISA.

Results

The IgG1 and IgG3 levels were elevated in the RA sera. The RA monocytes expressed more CD64 and cell surface-bound IgG than HC monocytes, and showed an impaired FcγR function as reflected by changes in IC-binding and decreased IC-stimulated TNFα secretion. These findings correlated significantly with different disease activity markers. Furthermore, sFcRs were elevated in the patient plasma, and sCD64 was specific for RA (compared with a reference group of patients with active psoriatic arthritis). Following treatment, immunoglobulins and sFcR levels were reduced, whereas membrane CD64 was only decreased in patients with good response to treatment.

Conclusions

Early RA patients display increased membrane and soluble CD64 and an impaired FcγR function correlating with joint disease activity. Beneficial responses of anti-rheumatic treatment in patients reduce CD64. These data suggest sCD64 as an important objective biomarker in RA.     

Localization of Cholesterol and Fatty Acid in a Model Lipid Membrane: A?Neutron Diffraction Approach

The intercellular lipid matrix of the skin’s stratum corneum serves to protect the body against desiccation and simultaneously limits the passage of drugs and other xenobiotics into the body. The matrix is made up of ceramides, free fatty acids, and cholesterol, which are organized as two coexisting crystalline lamellar phases. In studies reported here, we sought to use the technique of neutron diffraction, together with the device of isotopic (H/D) substitution, to determine the molecular architecture of the lamellar phase having a repeat distance of 53.9 ± 0.3 Å. Using hydrogenous samples as well as samples incorporating perdeuterated (C24:0) fatty acids and selectively deuterated cholesterol, the diffraction data obtained were used to construct neutron scattering length density profiles. By this means, the locations within the unit cell were determined for the cholesterol and fatty acids. The cholesterol headgroup was found to lie slightly inward from the unit cell boundary and the tail of the molecule located 6.2 ± 0.2 Å from the unit cell center. The fatty acid headgroups were located at the unit cell boundary with their acyl chains straddling the unit cell center. Based on these results, a molecular model is proposed for the arrangement of the lipids within the unit cell.     

Pharmacokinetics/Pharmacodynamics of Y‐700, A Novel Xanthine Oxidase Inhibitor,in Rats and Man

The pharmacokinetics and pharmacodynamics of a novel xanthine oxidase (XO) inhibitor, Y‐700, were evaluated in rats and healthy male volunteers. In a rat model of hyperuricemia, oral Y‐700 (0.3–10 mg/kg) showed a more potent and a longer‐lasting hypouricemic action than allopurinol. A single oral dosing of Y‐700 (5, 20 or 80 mg) to volunteers caused a dose‐dependent reduction of serum uric acid levels indicating close relationship to plasma concentrations of the compound. In addition, Y‐700 was hardly excreted in urine but mainly excreted in feces in rats and volunteers. These results suggested that Y‐700 is a new effective inhibitor of XO in rats and humans with high oral bioavailability being predominantly eliminated via the liver unlikely to allopurinol.     

Role of Mitochondria in Methamphetamine-Induced Dopaminergic Neurotoxicity: Involvement in Oxidative Stress,Neuroinflammation, and Pro-apoptosis—A Review

Methamphetamine (MA), an amphetamine-type psychostimulant, is associated with dopaminergic toxicity and has a high abuse potential. Numerous in vivo and in vitro studies have suggested that impaired mitochondria are critical in dopaminergic toxicity induced by MA. Mitochondria are important energy-producing organelles with dynamic nature. Evidence indicated that exposure to MA can disturb mitochondrial energetic metabolism by inhibiting the Krebs cycle and electron transport chain. Alterations in mitochondrial dynamic processes, including mitochondrial biogenesis, mitophagy, and fusion/fission, have recently been shown to contribute to dopaminergic toxicity induced by MA. Furthermore, it was demonstrated that MA-induced mitochondrial impairment enhances susceptibility to oxidative stress, pro-apoptosis, and neuroinflammation in a positive feedback loop. Protein kinase Cδ has emerged as a potential mediator between mitochondrial impairment and oxidative stress, pro-apoptosis, or neuroinflammation in MA neurotoxicity. Understanding the role and underlying mechanism of mitochondrial impairment could provide a molecular target to prevent or alleviate dopaminergic toxicity induced by MA.     

Beautiful,complicated—and intelligent? Novel aspects of the thigmonastic stamen movement in Loasaceae

In a recent study we investigated the complex mechanisms regulating the pollen release via thigmonastic stamen movement found exclusively in Loasaceae subfamily Loasoideae. We demonstrated that stamen movement is modulated by abiotic (light and temperature) as well as biotic stimuli (pollinator availability and visitation frequency). This is explained as a mechanism to adjust the rate of stamen movement and thus pollen dispensation to different environmental conditions in order to optimize pollen transfer. Stamen movement is rapid and thus a near-immediate response to pollinator visits. However, Loasaceae flowers also show a response to biotic stimuli on a longer time scale, by adjusting the duration of both the staminate and the carpellate phase of the anthesis. We here present two additional data sets on species not previously studied, underscoring the shortening of the staminate phase in the presence of pollinator visits vs. their absence and the shortening of the carpellate phase after pollination. Overall, the plant shows not only a rapid but an “intelligent” reaction to its environment in adjusting anthesis and pollen presentation to a range of factors. The physiological and morphological bases of the stamen movement are poorly understood. Our previous study showed that there is no direct spatial relationship between the place of stimulation in the flower and the stamen bundle activated. We here further show the morphological basis for stamen movement from a reflexed into an erect position: Only the basal part of the filament curves around the receptacle, while the upper part of the filament retains its shape. We hypothesize that the stimulus is transmitted over the entire receptacle and the place of reaction is determined by stamen maturity, not the location of the stimulus.