Influence of sequential oligopeptide carriers on the bioactive structure of conjugated epitopes: comparative study of the conformation of a Herpes simplex virus glycoprotein gD-1 epitope in the free and conjugated form, and protein "built-in" crystal structure

Synthetic carriers play an important role in immunogen presentation, due to their ability of inducing improved and specific responses to conjugated epitopes. Their influence on the bioactive conformation of the epitope, though admittedly crucial for relevant in vitro and in vivo applications, is difficult to evaluate, given the usual lack of information on the complex conformational features determined by the nature of the carrier and the mode of ligation. Using the Herpes simplex virus glycoprotein D-1 epitope (Leu(9)-Lys-Nle-Ala-Asp-Pro-Asn-Arg-Phe-Arg-Gly-Lys-Asp-Leu(22)) as a model, we have performed a detailed conformational analysis on the free epitope peptide in solution and on three constructs in which the epitope was conjugated to sequential oligopeptide carriers {Ac-[Lys-Aib-Gly](4)-OH (SOC(4))} (through either a thioether or an amide bond; Ac: acetyl) and polytuftsin oligomers {H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2) (T20)}, (through a thioether bond). The analysis of the epitope conformation in the parent protein, in carrier-conjugated and free form, suggests that the beta-turn structure of the -Asp(13)-Pro-Asn-Arg(16)- segment is highly conserved and independent of the epitope form. However, small conformational variations were observed at the C-terminal part of the epitope, depending on the nature of the carrier.     

Putrescine stimulates chemiosmotic ATP synthesis

Putrescine is a main polyamine found in animals, plants and microbes, but the molecular mechanism underlying its mode of action is still obscure. In vivo chlorophyll a fluorescence in tobacco leaf discs indicated that putrescine treatment affects the energization of the thylakoid membrane. Molecular dissection of the electron transport chain by biophysical and biochemical means provided new evidence that putrescine can play an important bioenergetic role acting as a cation and as a permeant natural buffer. We demonstrate that putrescine increases chemiosmotic ATP synthesis more than 70%. Also a regulation of the energy outcome by small changes in putrescine pool under the same photonic environment (i.e., photosynthetically active radiation) is shown. The proposed molecular mechanism has at least four conserved features: (i) presence of a membrane barrier, (ii) a proton-driven ATPase, (iii) a DeltapH and (iv) a pool of putrescine.     

Endothelial Ca2+ wavelets and the induction of myoendothelial feedback

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Mast cells and inflammation

Mast cells are well known for their role in allergic and anaphylactic reactions, as well as their involvement in acquired and innate immunity. Increasing evidence now implicates mast cells in inflammatory diseases where they are activated by non-allergic triggers, such as neuropeptides and cytokines, often exerting synergistic effects as in the case of IL-33 and neurotensin. Mast cells can also release pro-inflammatory mediators selectively without degranulation. In particular, IL-1 induces selective release of IL-6, while corticotropin-releasing hormone secreted under stress induces the release of vascular endothelial growth factor. Many inflammatory diseases involve mast cells in cross-talk with T cells, such as atopic dermatitis, psoriasis and multiple sclerosis, which all worsen by stress. How mast cell differential responses are regulated is still unresolved. Preliminary evidence suggests that mitochondrial function and dynamics control mast cell degranulation, but not selective release. Recent findings also indicate that mast cells have immunomodulatory properties. Understanding selective release of mediators could explain how mast cells participate in numerous diverse biologic processes, and how they exert both immunostimulatory and immunosuppressive actions. Unraveling selective mast cell secretion could also help develop unique mast cell inhibitors with novel therapeutic applications. This article is part of a Special Issue entitled: Mast cells in inflammation.     

Multiple factors influence calcium synchronization in arterial vasomotion

The intercellular synchronization of spontaneous calcium (Ca(2+)) oscillations in individual smooth muscle cells is a prerequisite for vasomotion. A detailed mathematical model of Ca(2+) dynamics in rat mesenteric arteries shows that a number of synchronizing and desynchronizing pathways may be involved. In particular, Ca(2+)-dependent phospholipase C, the intercellular diffusion of inositol trisphosphate (IP(3), and to a lesser extent Ca(2+)), IP(3) receptors, diacylglycerol-activated nonselective cation channels, and Ca(2+)-activated chloride channels can contribute to synchronization, whereas large-conductance Ca(2+)-activated potassium channels have a desynchronizing effect. Depending on the contractile state and agonist concentrations, different pathways become predominant, and can be revealed by carefully inhibiting the oscillatory component of their total activity. The phase shift between the Ca(2+) and membrane potential oscillations can change, and thus electrical coupling through gap junctions can mediate either synchronization or desynchronization. The effect of the endothelium is highly variable because it can simultaneously enhance the intercellular coupling and affect multiple smooth muscle cell components. Here, we outline a system of increased complexity and propose potential synchronization mechanisms that need to be experimentally tested.     

Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.     

GE-25-like immunoreactivity in the rat eye

This study aimed to investigate the presence and distribution of the chromogranin A-derived peptide GE-25 in the rat eye. The molecular form detected by the GE-25 antiserum was evaluated in the rat trigeminal ganglion, retina and remaining tissues of the rat eye by means of Western blots and the distribution pattern of GE-25-like immunoreactivity was studied in the rat eye and rat trigeminal ganglion by immunofluorescence. One single band of approximately 70kDa was stained in the trigeminal ganglion and retina which represents the uncleaved intact chromogranin A indicating that the proteolytic processing of chromogranin A to GE-25 is limited in these tissues. Sparse GE-25-like immunoreactive nerve fibers were visualized in the corneal stroma, at the limbus around blood vessels, in the sphincter and dilator muscle and stroma of the iris, in the stroma of the ciliary body and ciliary processes and in the stroma and around blood vessels in the choroid. This distribution pattern is characteristic for neuropeptides whereas the presence of immunoreactivity in the corneal endothelium and in Müller glia in the retina is atypical. GE-25-like immunoreactivity was found in small to medium-sized ganglion cells in the rat trigeminal ganglion clearly indicating that the nerve fibers in the rat eye are of sensory origin. The colocalization of GE-25-immunoreactivity with SP-immunoreactivity in the rat ciliary body is in agreement with the presumption of the sensory nature of the innervation of the anterior segment of the eye by GE-25.     

Quercetin is more effective than cromolyn in blocking human mast cell cytokine release and inhibits contact dermatitis and photosensitivity in humans

Mast cells are immune cells critical in the pathogenesis of allergic, but also inflammatory and autoimmune diseases through release of many pro-inflammatory cytokines such as IL-8 and TNF. Contact dermatitis and photosensitivity are skin conditions that involve non-immune triggers such as substance P (SP), and do not respond to conventional treatment. Inhibition of mast cell cytokine release could be effective therapy for such diseases. Unfortunately, disodium cromoglycate (cromolyn), the only compound marketed as a mast cell "stabilizer", is not particularly effective in blocking human mast cells. Instead, flavonoids are potent anti-oxidant and anti-inflammatory compounds with mast cell inhibitory actions. Here, we first compared the flavonoid quercetin (Que) and cromolyn on cultured human mast cells. Que and cromolyn (100 μM) can effectively inhibit secretion of histamine and PGD(2). Que and cromolyn also inhibit histamine, leukotrienes and PGD(2) from primary human cord blood-derived cultured mast cells (hCBMCs) stimulated by IgE/Anti-IgE. However, Que is more effective than cromolyn in inhibiting IL-8 and TNF release from LAD2 mast cells stimulated by SP. Moreover, Que reduces IL-6 release from hCBMCs in a dose-dependent manner. Que inhibits cytosolic calcium level increase and NF-kappa B activation. Interestingly, Que is effective prophylactically, while cromolyn must be added together with the trigger or it rapidly loses its effect. In two pilot, open-label, clinical trials, Que significantly decreased contact dermatitis and photosensitivity, skin conditions that do not respond to conventional treatment. In summary, Que is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases, especially in formulations that permit more sufficient oral absorption.