Hesperidin inhibits expression of hypoxia inducible factor-1 alpha and inflammatory cytokine production from mast cells

The citrus unshiu peel has been used traditionally as a medicine to improve bronchial and asthmatic conditions or cardiac and blood circulation in Korea, China, and Japan. Here, we report the effects of citrus unshiu peel water extract (CPWE) on the phorbol myristate acetate (PMA) + calcium ionophore A23187-induced hypoxia-inducible factor-1α (HIF-1α) activation and inflammatory cytokine production from the human mast cell line, HMC-1 cells. We compared CPWE with hesperidin, a common constituent of citrus unshiu. CPWE and hesperidin inhibited the PMA + A23187-induced HIF-1α expression and the subsequent production of vascular endothelial growth factor (VEGF). In addition, CPWE suppressed PMA + A23187-induced phosphorylation of the extracellular signal-regulated kinase (ERK). We also show that the increased cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α level was significantly inhibited by treatment of CPWE or hesperidin. In the present study, we report that CPWE and hesperidin are inhibitors of HIF-1α and cytokines on the mast cell-mediated inflammatory responses.     

Characterization of α-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

α-mannosidase from Erythrina indica seeds is a Zn²⁺ dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 °C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol^− 1. N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3–8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of α-helical structure in the enzyme. α-Mannosidase from E indica exhibits immunological identity with α-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with α-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of α-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

Leptin attenuates gene expression for renal 25-hydroxyvitamin D3-1alpha-hydroxylase in mice via the long form of the leptin receptor

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We previously showed that leptin administration to leptin-deficient obese (ob/ob) mice suppressed mRNA expression and activity of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). In leptin receptor-deficient (db/db) mice, we presently examined whether leptin affects 1alpha-hydroxylase expression in renal tubules through the active form of the leptin receptor (ObRb). Elevated serum concentrations of calcium and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in untreated ob/ob mice showed sharp reduction with leptin administration (4 mg/kg, i.p. every 12h for 2 days); no such reduction of elevation occurred in db/db mice. ObRb mRNA was expressed in kidney, brain, fat, lung, and bone in wild-type and ob/ob mice, but not db/db mice. The ob/ob and db/db mice showed large increases in renal 1alpha-hydroxylase mRNA expression and activity. Leptin administration (4 mg/kg) completely abrogated these increases in ob/ob but not db/db mice. Renal 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA synthesis also was greatly elevated in ob/ob and db/db mice; excesses decreased significantly with leptin administration in ob/ob mice, but increased in db/db mice. Renal tubular cells in primary culture expressed mRNAs including proximal tubules markers (1alpha-hydroxylase and megalin), parathyroid hormone receptor, and vitamin D receptor. Calcitonin receptor mRNA, synthesized mainly in distal tubules, was scant, indicating that most cultured cells were from proximal tubules. Cells did not express ObRb mRNA. Forskolin exposure at 10(-6)M for 3 or 6h significantly increased 1alpha-hydroxylase mRNA. Leptin at 10(-6)M did not change mRNA expression in either presence or absence of forskolin. Accordingly, leptin attenuates renal 1alpha-hydroxylase gene expression through ObRb. Furthermore, leptin appears to act indirectly on renal proximal tubules to regulate 1alpha-hydroxylase gene expression.     

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A²) and SP-A2 (1A⁰), and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A⁰) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A²) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.     

Structure of the C-terminal domain of the pro-apoptotic protein Hrk and its interaction with model membranes

The protein harakiri (Hrk) is a pro-apoptotic BH3-only protein which belongs to the Bcl-2 family. Hrk appears associated to the mitochondrial outer membrane, apparently by a putative transmembrane domain, where it exerts its function. In this work we have identified a 27mer peptide supposed to be the putative membrane domain of the protein at the C-terminal region, and used infrared and fluorescence spectroscopies to study its secondary structure as well as to characterize its effect on the physical properties of phospholipid model membranes. The results presented here showed that the C-terminal region of Hrk adopts a predominantly α-helical structure whose proportion and destabilization capability varied depending on phospholipid composition. Moreover it was found that the orientation of the α-helical component of this C-terminal Hrk peptide was nearly perpendicular to the plane of the membrane. These results indicate that this domain is able of inserting into membranes, where it adopts a transmembrane α-helical structure as well as it considerably perturbs the physical properties of the membrane.     

Characterization of the conformational and orientational dynamics of ganglioside GM1 in a dipalmitoylphosphatidylcholine bilayer by molecular dynamics simulations

The structure and dynamics of a single GM1 (Gal5-β1,3-GalNAc4-β1,4-(NeuAc3-α2,3)-Gal2-β1,4-Glc1-β1,1-Cer) embedded in a DPPC bilayer have been studied by MD simulations. Eleven simulations, each of 10 ns productive run, were performed with different initial conformations of GM1. Simulations of GM1-Os in water and of a DPPC bilayer were also performed to delineate the effects of the bilayer and GM1 on the conformational and orientational dynamics of each other. The conformation of the GM1 headgroup observed in the simulations is in agreement with those reported in literature; but the headgroup is restricted when embedded in the bilayer. NeuAc3 is the outermost saccharide towards the water phase. Glc1 and Gal2 prefer a parallel, and NeuAc3, GalNac4 and Gal5 prefer a perpendicular, orientation with respect to the bilayer normal. The overall characteristics of the bilayer are not affected by the presence of GM1; however, GM1 does influence the DPPC molecules in its immediate vicinity. The implications of these observations on the specific recognition and binding of GM1 embedded in a lipid bilayer by exogenous proteins as well as proteins embedded in lipids have been discussed.