Impact of sphingomyelin acyl chain heterogeneity upon properties of raft-like membranes.

Sphingomyelin (SM) is a main component of lipid rafts and characteristic of abundance of long and saturated acyl chains. Recently, we reported that fluorescence-labeled lipids including C16:0 and C18:0SMs retained membrane behaviors of inherent lipids. Here, we newly prepared fluorescent SMs with longer acyl chains, C22:0 and C24:1, for observing their partition and diffusion in SM/cholesterol (chol)/dioleoylphosphatidylcholine (DOPC) bilayers. Although fluorescent C24:1SM underwent a uniform distribution between ordered (Lo) and disordered (Ld) phases, other fluorescent SMs with saturated acyl chains were preferentially distributed in the Lo phase. Interestingly, when the acyl chains of fluorescent and membrane SMs are different, distribution of fluorescent SM to the Lo phase was reduced compared to when the acyl chains are the same. This tendency was also observed for C16:0SM/C22:0SM/chol/DOPC quaternary bilayers, where the minor SM was more excluded out of the Lo phase than the major SM. We also found that the coexistence of SMs induces SM efflux out of the Lo phase and simultaneous DOPC influx to the Lo phase, consequently reducing the difference in fluidity between the two phases. These results suggest that physicochemical properties of lipid rafts are regulated by the acyl chain heterogeneity of SMs.

The phosphodiesterase 5 inhibitor tadalafil has renoprotective effects in a rat model of chronic kidney disease.

Phosphodiesterase 5 inhibitors are widely used to treat erectile dysfunction and lower urinary tract symptoms with benign prostatic hyperplasia. Recent studies have indicated the renoprotective effects of this class of compounds. Whether renoprotection depends on blood pressure reduction remains controversial. In this study, we investigated the renoprotective effects of the phosphodiesterase 5 inhibitor, tadalafil, in a rat model of high-salt induced kidney injury with hypertension. Dahl salt-sensitive rats were fed a normal diet, high-salt (8% sodium chloride) diet, or high-salt diet with oral administration of either low- or high-dose tadalafil (1 and 10 mg kg-1  day-1 , respectively). Serum creatinine, urinary protein, and blood pressure were measured at baseline and after 8 weeks, at which point the rats were examined for glomerular injury and fibrosis. PAI1 mRNA levels were also evaluated. After 8 weeks, blood pressure, serum creatinine, and urinary protein levels were significantly higher in the high-salt group than those in the normal-salt group. Serum creatinine and urinary protein were significantly lower in both tadalafil groups than those in the high-salt group, while only high-dose tadalafil affected blood pressure. In addition, glomerulosclerosis and α-smooth muscle actin expression significantly decreased in both tadalafil treatment groups. PAI1 mRNA increased significantly in the high-salt group but decreased in both tadalafil-treated groups. Our results indicated that both low- and high-dose tadalafil prevented fibrosis and glomerular injury in a chronic kidney disease rat model. Mechanistically, these effects may be associated with PAI1 expression and glomerular structure protection.

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins.

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28-58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.

Generation of transgenic rice with reduced content of major and novel high molecular weight allergens.

BACKGROUND: Rice seed proteins contain antigens that provoke allergic responses in some individuals with food allergy, particularly in those with cereal allergy, and these antigens can elicit clinical symptoms such as eczema and dermatitis. We previously generated transgenic rice with reduced accumulation of the three major allergens, which dramatically reduced the level of IgE binding from patients' sera. However, the transgenic rice still possesses allergenic reactivity. Recently, two globulin-like proteins were identified as candidates of novel high molecular weight (HMW) IgE-binding proteins that cause rice allergy. RESULTS: We identified a glucosidase family encoded by four genes as novel HMW rice allergens based on IgE antibody reactivity from individuals with allergy to rice. To further reduce allergenicity, we generated transgenic rice with reduced accumulation of these HMW allergens. We crossed the rice with reduced HMW allergens and with reduced major allergens, and all major and HMW allergens were substantially reduced in the progeny of the crossed rice. Allergen suppression did not significantly alter accumulation patterns of seed storage proteins and protein folding enzymes. The sera of a portion of patients showed low IgE-binding to the crossed line, suggesting that the crossed line is effective for a portion of patients who are allergic to proteins other than major allergens. CONCLUSIONS: The transgenic rice with reduced levels of all major and HMW allergens is thought to be an option for a portion of allergy patients with hypersensitive responses to various kinds of rice allergens.

Allergenic potential of rice-pollen proteins: expression, immuno-cross reactivity and IgE-binding.

Pollen proteins from several grass species have been identified and characterized as causative allergens in grass pollinosis. In contrast, allergenic potential of pollen proteins from rice, which belongs to the same Poaceae family, has not well been investigated, despite that a few clinical cases have been reported on rice-pollen allergy. In this study, to characterize expression and allergenic potential of pollen proteins from rice (Oryza sativa, ssp. japonica), rice putative proteins for ß-expansin (EXP), a Ca(2+)-binding protein (CBP)/polcalcin, extensin (EXT), profilin (PRF) and polygalacturonase (PGA) retrieved from a rice complete cDNA database were prepared as recombinant proteins, and the antibodies to these recombinant proteins were obtained. Immuno-blotting and immuno-histological analyses showed that rice putative EXP, EXT and PGA were expressed abundantly in anther tissue and pollen granules and immuno-cross reactive with pollen proteins from timothy grass. ELISA and immuno-dot blotting analyses using serum specimens from allergic patients showed that majority of the specimens was positive in the IgE-binding to EXP and EXT, but weakly to PGA and almost negative to PRF. EXP and EXT were suggested to be potentially allergenic in the rice-pollen allergy as well as the grass pollinosis.

Generation of transgenic rice lines with reduced contents of multiple potential allergens using a null mutant in combination with an RNA silencing method.

Rice seed proteins are known to be a causative antigen in some patients with food allergy, especially cereal allergy, with clinical symptoms such as eczema and dermatitis. The α-amylase/trypsin inhibitors (14-16 kDa), α-globulin (26 kDa) and ß-glyoxalase I (33 kDa) are regarded as major potential allergens of rice (Oryza sativa L.) seed based on specific recognition by serum IgE from allergy patients. In order to suppress the production of these major allergens in rice grains, a mutant in the 'Koshihikari' background lacking the 26 kDa allergen (GbN-1) was used as a host for RNA silencing. A binary vector harboring two RNA interference (RNAi) gene cassettes for suppression of 14-16 kDa and 33 kDa allergens driven by the 13 kDa and 10 kDa prolamin endosperm-specific promoters, respectively, was introduced into the GbN-1 genome by Agrobacterium-mediated transformation. In the most promising transgenic line, the content of the three potential allergens was remarkably reduced to a very faint level without a change in seed phenotype. IgE binding of 15 patients' sera to the transgenic rice seed mostly deficient in the three major allergens was on average only about 10% that of the control wild-type rice, suggesting that these three accounted for the great majority of rice seed causative allergens recognized by patients' IgE and that the sequential allergen deletion/reduction strategy works in the development of hypo-allergenic rice lines.

Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis of hyaluronan oligosaccharides.

A new method is presented for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronan (HA) with bacterial hyaluronidase (E.C. 4.2.2.1, from Streptomyces hyalurolyticus) using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Mixtures containing HA oligosaccharides of tetrasaccharide (4-mer)-34-mer were analyzed using this method. The carboxyl groups of the glucuronate residues in the prepared HA oligomers, were modified as the acidic form (-COOH), sodium salts (-COONa), organic ammonium salts, or methylesters before MALDI-TOFMS measurement. Among these samples, the methylester form of glucuronate residues in HA oligosaccharides, prepared by methylation using trimethylsilyl diazomethane, afforded high sensitivity for spectra. This simple modification method for carboxyl group methylation of acidic polysaccharides [Hirano et al., Carbohydr. Res., 340, (2005) 2297-2304] provides samples suitable for MALDI-TOF mass spectrometric analysis throughout a significantly enhanced range of masses.

Preparation of the methyl ester of hyaluronan and its enzymatic degradation.

A methyl ester of hyaluronan in which the carboxyl groups were fully esterified was prepared using trimethylsilyl diazomethane. This derivative, while not depolymerized by hyaluronan lyases or hyaluronan hydrolases, was a substrate for both chondroitin ACI lyase (EC 4.2.2.5) from Flavobacterium heparinum and chondroitin ACII lyase (EC 4.2.2.5) from Arthrobacter aurescens. The major product isolated in these depolymerization reactions was methyl alpha-L-threo-hex-4-enepyranosyluronate-(1-->3)-2-acetamido-2-deoxy-alpha,beta-D-glucopyranoside as determined by 1H NMR spectroscopy and MALDITOF mass spectrometry.