Determination of tetrabromobisphenol A in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A method for the determination of tetrabromobisphenol A (TBBPA) in human serum utilizing solid-phase extractions (SPEs) and liquid chromatography (LC) with electrospray ionization tandem MS (MS/MS) has been developed. After purification and concentration of TBBPA using consecutive SPEs on reversed-phase and normal-phase cartridges, the serum sample was subjected to LC. TBBPA was separated on a C18 reversed-phase column by gradient elution with a mixture of water, methanol, and acetonitrile as the mobile phase, and then detected with electrospray ionization MS/MS in negative ion mode. 13C12-TBBPA was suitable as an internal standard for the reproducible determination of TBBPA in human serum samples (5 g). The method has been validated in TBBPA concentration range of 5-100 pg per g serum, and the recoveries in the concentration range were higher than 83.3%. The repeatabilities of the proposed method of non-spiked control serum (6.3 pg per g serum) and spiked serum (added 5-100 pg per g serum) were within 10.0% as relative standard deviations. The limit of quantification (LOQ) for TBBPA was 4.1 pg per g serum, which was corresponded to 0.63 fmol on column.     

Antigen presentation by Peyer's patch cells can induce both Th1- and Th2-type responses depending on antigen dosage,but a different cytokine response pattern from that of spleen cells

The Th1 and Th2 preference induced by cells from the Peyer's patch (PP) and spleen (SPL) with various doses of an antigen was examined. The same splenic T cell receptor-transgenic CD4+ T cells were first incubated with PP or SPL cells in the presence of various doses of an antigen, and the cytokine response was observed after secondary stimulation. A Th2-type pattern was only obtained for primary stimulation at 10 microM of the antigen with PP cells, whereas a Th1 pattern was induced at both higher and lower concentrations. SPL cells in the presence of 0.1 to 1 microM of the antigen induced the secretion of Th2-type cytokines. Ten and 100 microM of the antigen plus SPL cells did not induce the release of a large quantity of cytokines. PP cells induced a different cytokine pattern at the antigen concentration that induced a similar level of T cell proliferation with SPL cells. Our findings suggest that the antigen-dose dependent development of Th1/Th2 cells is differentially modulated by the antigen-presentation function of cells in PP and SPL.     

Epitope analysis of antibodies in Japanese to human cytomegalovirus phosphoprotein 150 with synthetic peptides

Serological detection of antibodies specific to human cytomegalovirus (HCMV) is not reliable because the assay uses the whole HCMV protein fraction. Antigenic materials composed of well-characterized viral proteins are being tried for serodiagnosis in Europe. Epitopes of antibodies to HCMV phosphoprotein 150 (pp150) encoded by UL32 in Japanese individuals were investigated for comparison with the results in Europe. The major epitopes on amino acid residues 496 to 652 of HCMV pp150 were identified and the detection of antibodies with an enzyme-linked immunosorbent assay (ELISA) of synthetic peptides against the main epitopes was established. Fifteen seropositive and five seronegative serum samples for the epitope mapping and 131 seropositive and 50 seronegative samples for ELISA were investigated. Overlapping 15-mer peptides moving by two amino acids through V496-H652 were synthesized. The main epitope regions were V508-D530, L526-Q544, S536-D554, T616-G634, S624-P642, and L632-H652. When each peptide was conjugated with bovine serum albumin for ELISA, 80.9% of the seropositive samples were judged to be positive. The results of this study are the same as those for European sera, so the antigenic materials developed in Europe might be used to replace the whole HCMV protein fraction in Japanese.     

Characterization of five phyllosphere bacteria isolated from Rosa rugosa leaves,and their phenotypic and metabolic properties

Five gram-negative bacteria, all of which were Enterobacteriaceae, were isolated from the phyllosphere of green or senescing leaves of Rosa rugosa, and their phenotypic and physiological characteristics were examined. Partial 16S rDNA sequences led to identification of these isolates as Pantoea agglomerans, Klebsiella terrigena, Erwinia rhapontici, and two strains of Rahnella aquatilis. Interestingly, these phyllosphere bacteria had certain phenotypic and physiological convergences, while they showed their own metabolic properties toward phenolic compounds of plant origin. In particular, the two Ra. aquatilis isolates from the green leaves had a substrate-inducible gallate decarboxylase activity in the resting cells that had been cultured in 1 mM gallic acid- or protocatechuic acid-containing medium. The other three isolates from the senescing leaves did not have this enzyme activity. Simple phenolics that the Ra. aquatilis decarboxylatively produced from benzoic acid derivatives had better antimicrobial activities than those of the substrates.     

Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation

The therapeutic mechanisms of lithium for treating bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults. Here, we studied neuroprotective effects of lithium against excitotoxicity in cultured cerebral cortical neurons. Glutamate-induced excitotoxicity in cortical neurons was exclusively mediated by NMDA receptors. Pre-treatment of cortical neurons with LiCl time-dependently suppressed excitotoxicity with maximal protection after 6 days of pre-treatment. Significant protection was observed at the therapeutic and subtherapeutic concentration of 0.2-1.6 mm LiCl with almost complete protection at 1 mM. Neuroprotection was also elicited by valproate, another major mood-stabilizer. The neuroprotective effects of lithium coincided with inhibition of NMDA receptor-mediated calcium influx. Lithium pre-treatment did not alter total protein levels of NR1, NR2A and NR2B subunits of NMDA receptors. However, it did markedly reduce the level of NR2B phosphorylation at Tyr1472 and this was temporally associated with its neuroprotective effect. Because NR2B tyrosine phosphorylation has been positively correlated with NMDA receptor-mediated synaptic activity and excitotoxicity, the suppression of NR2B phosphorylation by lithium is likely to result in the inactivation of NMDA receptors and contributes to neuroprotection against excitotoxicity. This action could also be relevant to its clinical efficacy for bipolar patients.     

Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver

The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 (P<0.05) along with a 2.9-fold elevation in CA concentration (P<0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the cholesterol 7alpha-hydroxylase, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase and 5beta-cholestane-3alpha,7alpha,12alpha-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and enzyme mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, bile acid biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters.     

Physical association of beta 2 integrin with GPI-80, a novel glycosylphosphatidylinositol-anchored protein with potential for regulating adhesion and migration

We recently found a novel GPI-anchored protein, GPI-80, on human phagocytes that may regulate leukocyte adherence, locomotion, and extravasation. To clarify the mechanisms by which GPI-80 functions on leukocytes, we explored the possibility of its physical association with beta 2 integrin which is important for leukocyte adherence, locomotion, and extravasation. beta 2 integrin, detected by anti-CD18 mAb, was coprecipitated with GPI-80 from human neutrophil lysates by a mAb to GPI-80. In addition, GPI-80 was immunoprecipitated from human neutrophil lysates by anti-human CD18 mAb. These results clearly show that GPI-80 is physically associated with beta 2 integrin in human neutrophils.     

Crystallization and preliminary x-ray studies of the Fab fragment from a humanized version of the mouse anti-human fas antibody HFE7a

A humanized version of the apoptosis-inducing mouse anti-human Fas monoclonal antibody, HFE7A, is under further development for the treatment of autoimmune diseases such as rheumatoid arthritis. We have crystallized the antigen-binding fragment (Fab) of the humanized HFE7A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 54.4 A, b = 82.7 A, c = 104.9 A and contain one Fab molecule in the asymmetric unit. X-ray diffraction data were collected to 2.8 A resolution.     

Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin

This study was designed to clarify the mechanisms of hypocholesterolemic action of beta-lactoglobuline tryptic hydrolysate (LTH) and to identify the novel hypocholesterolemic peptide derived from LTH by screening using Caco-2 cells and animal studies. Serum and liver cholesterol levels were significantly lower in rats fed LTH than in those fed casein tryptic hydrolysate (CTH). The present study suggests that the inhibition of micellar solubility of cholesterol which causes the suppression of cholesterol absorption by a direct interaction between cholesterol mixed micelles, and LTH in the jejunal epithelia is part of the mechanism underlying the hypocholesterolemic action of LTH. Though no one could trace the hypocholesterolemic peptide to any protein origin, we identified, for the first time, a novel hypocholesterolemic peptide, Ile-Ile-Ala-Glu-Lys (IIAEK). Surprisingly, the present study provides the first direct evidence that a new hypocholesterolemic peptide derived from beta-lactoglobuline can powerfully influence serum cholesterol levels and exhibit a greater hypocholesterolemic activity in comparison with that of medicine, beta-sitosterol, in animal studies.     

Multiple molecular forms of glucagon and insulin in the kaluga sturgeon, Huso dauricus

Five molecular forms of glucagon and two molecular forms of insulin were characterized from the kaluga sturgeon. Substitutions occurred at two to thirteen internal amino acid residues among the five molecular forms of glucagons, indicating that these glucagons were encoded by five distinct genes. The amino acid sequences of two insulins from the kaluga sturgeon were identical to those of paddlefish insulin-II and Russian sturgeon insulin except that kaluga sturgeon insulin-I had an extension of five residues at the B-chain N-terminus. This is the first demonstration that more than two molecular forms of glucagon have been characterized from a single animal species.