The review focuses on the unique spectral features of pyrene that can be utilized to investigate protein structure and conformation. Pyrene is a fluorescent probe that can be attached covalently to protein side chains, such as sulfhydryl groups. The spectral features of pyrene are exquisitely sensitive to the microenvironment of the probe: it exhibits an ensemble of monomer fluorescence emission peaks that report on the polarity of the probe microenvironment, and an additional band at longer wavelengths, the appearance of which reflects the presence of another pyrene molecule in spatial proximity (~10 Å). Its high extinction coefficient allows us to study labeled proteins in solution at physiologically relevant concentrations. The environmentally- and spatially-sensitive features of pyrene allow monitoring protein conformation, conformational changes, protein folding and unfolding, protein-protein, protein-lipid and protein-membrane interactions.

Autism spectrum disorders (ASDs) affect as many as 1 in 45 children and are characterized by deficits in sociability and communication, as well as stereotypic movements. Many children also show severe anxiety. The lack of distinct pathogenesis and reliable biomarkers hampers the development of effective treatments. As a result, most children with ASD are prescribed psychopharmacologic agents that do not address the core symptoms of ASD. Autoantibodies against brain epitopes in mothers of children with ASD and many such children strongly correlate with allergic symptoms and indicate an aberrant immune response, as well as disruption of the blood–brain barrier (BBB). Recent epidemiological studies have shown a strong statistical correlation between risk for ASD and either maternal or infantile atopic diseases, such as asthma, eczema, food allergies and food intolerance, all of which involve activation of mast cells (MCs). These unique tissue immune cells are located perivascularly in all tissues, including the thalamus and hypothalamus, which regulate emotions. MC-derived inflammatory and vasoactive mediators increase BBB permeability. Expression of the inflammatory molecules interleukin (IL-1β), IL-6, 1 L-17 and tumor necrosis factor (TNF) is increased in the brain, cerebrospinal fluid and serum of some patients with ASD, while NF-kB is activated in brain samples and stimulated peripheral blood immune cells of other patients; however, these molecules are not specific. Instead the peptide neurotensin is uniquely elevated in the serum of children with ASD, as is corticotropin-releasing hormone, secreted from the hypothalamus under stress. Both peptides trigger MC to release IL-6 and TNF, which in turn, stimulate microglia proliferation and activation, leading to disruption of neuronal connectivity. MC-derived IL-6 and TGFβ induce maturation of Th17 cells and MCs also secrete IL-17, which is increased in ASD. Serum IL-6 and TNF may define an ASD subgroup that benefits most from treatment with the natural flavonoid luteolin. Atopic diseases may create a phenotype susceptible to ASD and formulations targeting focal inflammation of the brain could have great promise in the treatment of ASD.

Plasma HDLs are thought to protect against atherosclerosis via mechanisms related, in part, to reverse cholesterol transport (RCT) ( 1-3 ). This process involves removal of excess cholesterol from arterial foam-cells (i.e., cholesterol effl ux), followed by the transport of cholesterol to the liver for excretion in feces. Over-expression of apolipoprotein (apo) A-I, the major protein of HDL, in mice enhances RCT, inhibits atherosclerosis development, and stimulates regression of plaque lesions ( 4-8 ). Similar effects can be obtained upon infusion of apoA-I-phospholipid complexes in mice and humans, suggesting HDL may be useful therapeutically ( 9-11 ).Small peptides based on HDL apolipoproteins are being developed as alternatives to full-length recombinant proteins for therapeutic use (12)(13)(14). The mechanisms by which apolipoprotein mimetic peptides reduce atherosclerosis are not fully understood. The most widely studied 4F peptide reduces atherosclerosis in animal models via mechanisms related to binding of oxidized lipid ( 12,15 ). The latter anti-oxidant activity greatly exceeds that of apoA-I ( 16 ). The 18A/4F peptides can stimulate cholesterol effl ux from cells, albeit with far weaker activity Abstract Here, we report the creation of a single-helix peptide (ATI-5261) that stimulates cellular cholesterol effl ux with K m molar effi ciency approximating native apolipoproteins. Anti-atherosclerosis activity of ATI-5261 was evaluated in LDLR ؊ / ؊ and apolipoprotein (apo)E ؊ / ؊ mice ‫ف‬ 5-7 months of age, following 13-18 weeks on a high-fat Western diet (HFWD). Treatment of fat-fed LDLR؊ / ؊ mice with daily intraperitoneal injections of ATI-5261 (30 mg/kg) for 6 weeks reduced atherosclerosis by 30%, as judged by lesion area covering the aorta (7.9 ± 2 vs.11.3 ± 2.5% control, P = 0.011) and lipid-content of aortic sinus plaque (25 ± 5.8 vs. 33 ± 4.9% control, P = 0.014). In apoE ؊ / ؊ mice, the peptide administered 30 mg/kg ip on alternate days for 6 weeks reduced atherosclerosis by ‫ف‬ 45% (lesion area = 15 ± 7 vs. 25 ± 8% control, P = 0.00016; plaque lipid-content = 20 ± 6 vs. 32 ± 8% control, P < 0.0001). Similar reductions in atherosclerosis were achieved using ATI-5261:POPC complexes. Single intraperitoneal injection of ATI-5261 increased reverse cholesterol transport from macrophage foam-cells to feces over 24-48 h. In summary, relatively short-term treatment of mice with the potent cholesterol effl ux peptide ATI-5261 reduced substantial atherosclerosis. This was achieved using an L-amino acid peptide, in the presence of severe hypercholesterolemia/ HFWD, and did not require daily injections or formulation with phospholipids when administered via intraperitoneal

Background Mast cells (MCs) are hemopoietic cells that mature in tissues and are involved in allergy, immunity and inflammation by secreting multiple mediators. The natural flavone luteolin (lut) has anti-inflammatory actions and inhibits human MCs. Objective To investigate the ability of lut, and its novel structural analog 3’,4’,5,7-tetramethoxyluteolin (methlut), to inhibit human MCs mediator expression and release in vitro and in vivo. Methods Human LAD2 cells and primary human umbilical cord-blood derived cultured MC (hCBMCs) were stimulated by substance P (SP) or IgE/anti-IgE with or without pre-incubation with lut, methlut or cromolyn (1–100 μM) for 2 or 24 hr following which a mediator secretion was measured. The effect of the compound on MC intracellular calcium levels and NF-κB activation was also investigated. Pretreatment with methlut was also studied in mice passively sensitized with dinotrophenol-human serum albumin (DNP-HSA) and challenged intradermally. Results Methlut is a more potent inhibitor than lut or cromolyn for beta-hexosaminidase (β-hex) and histamine secretion from LAD2 cells stimulated by either SP or IgE/anti-IgE, but only methlut and lut significantly inhibit preformed tumor necrosis factor (TNF) secretion. Methlut is also a more potent inhibitor than lut of de novo synthesized TNF from LAD2, and of chemokine (C-C motif) ligand 2 (CCL2) from hCBMCs. The mechanism of action from methlut may be due to its ability to inhibit intracellular calcium increase, as well as NF-κB induction at both the transcriptional and translational levels in LAD2 cells stimulated by SP without affecting cell viability. Treatment (ip) with methlut significantly decreases skin vascular permeability of Evans blue in mice passively sensitized to DNP-HAS and challenged intradermaly. Conclusion Methlut is a promising MC inhibitor for the treatment of allergic and inflammatory conditions.