Hydroxylation of para-chlorotoluene by model complexes of cytochrome P-450

Hydroxylation of p-chlorotoluene with heminthiol complexes, Fenton's system and Udenfriend's system was studied and the complexes assessed as models of cytochrome P-450 monooxygenases. Five species of possible hydroxylation products of p-chlorotoluene, namely, p-chlorobenzyl alcohol, 2-chloro-5-methylphenol, p-chlorobenzaldehyde, 4-chloro-2-methylphenol and 5-chloro-2-methylphenol, were studied using high performance liquid chromatography. The oxidation reactions were characterized by the yields of hydroxylation products and the product ratio. The system consisting of hemin and cysteine ethyl ester as well as Udenfriend's system gave relatively high hydroxylation yields and the former only induced a methyl migration during hydroxylation (methyl NIH shift). However, neither Fenton's nor Udenfriend's systems induced a methyl NIH shift. The hemin-thiol complex is thus concluded to be a good chemical model of cytochrome P-450 monooxygenases.     

Recognition by TNF-alpha of the GPI-anchor glycan induces apoptosis of U937 cells

Tumor necrosis factor-alpha (TNF-alpha) binds to TNF-alpha receptors (TNFR) to produce a hexameric (TNF-alpha)(3)-(TNFR)(3) structure that stimulates apoptosis. We found by using ELISA that TNF-alpha binds to the glycosylphosphatidylinositol (GPI) anchor glycans of carcinoembryonic antigen, human placental alkaline phosphatase (hAP), and Tamm-Horsfall glycoprotein. These binding abilities were inhibited by 10(-6)M mannose-6-phosphate. Treatment of hAP with mild acid and phosphatase, which releases the N-acetylglucosamine (GlcNAc) beta1 -->phosphate-->6 residue from the GPI-anchor glycan of hAP, abrogated the binding of TNF-alpha to hAP. Thus, TNF-alpha binds to the GlcNAcbeta1-->phosphate-->6Man residue in GPI-anchor glycans. To investigate whether the carbohydrate-binding ability of TNF-alpha is related to its physiological functions, human lymphoma U937 cells were used. TNF-alpha stimulates U937 cell apoptosis in a dose-dependent manner and the presence of mannose-6-phosphate inhibited this. TNF-alpha-dependent tyrosine phosphorylation of several proteins in U937 cells was also diminished by mannose-6-phosphate. Phosphatidylinositol-specific phospholipase C-pretreatment also inhibited this tyrosine phosphorylation. These data suggest that TNF-alpha stimulates U937 cell apoptosis by forming a high-affinity nanomeric (TNF-alpha)(3)-(TNFR)(3)-(GPI-anchored glycan)(3) complex. The GPI-anchored glycoprotein involved remains to be identified.     

A retaining endo-beta-mannosidase from a dicot plant, cabbage

An endo-beta-mannosidase [EC 3.2.1.152, glycoside hydrolase family 2], which hydrolyzes the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, has been found in plant tissues [Ishimizu, T., Sasaki, A., Okutani, S., Maeda, M., Yamagishi, M., and Hase, S. (2004) J. Biol. Chem. 279, 38555-38562]. So far, this glycosidase has been purified only from a monocot plant, a lily. Here, an endo-beta-mannosidase was purified from a dicot plant, cabbage (Brassica oleracea), and characterized. The cabbage endo-beta-mannosidase consists of four polypeptides. These four polypeptides are encoded by a single gene, whose nucleotide sequence is homologous to those of the lily and Arabidopsis endo-beta-mannosidase genes. 1H NMR analysis of the stereochemistry of the hydrolysis of pyridylaminated (PA) Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc showed that the cabbage endo-beta-mannosidase is a retaining glycoside hydrolase, as are other glycoside hydrolase family 2 enzymes. The enzymatic characteristics, including substrate specificity, of the cabbage enzyme are very similar to those of the lily enzyme. These endo-beta-mannosidases specifically act on Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0 to 2). These results suggest that the endo-beta-mannosidase is present in at least the angiosperms, and has common roles, such as the degradation of N-glycans.     

Micellization of conjugated chenodeoxy- and ursodeoxycholates and solubilization of cholesterol into their micelles: comparison with other four conjugated bile salts species

Micelle formations of sodium glyco- and taurochenodeoxycholate (NaGCDC and NaTCDC) and sodium glyco- and tauroursodeoxycholates (NaGUDC and NaTUDC) was studied at 308.2 K for their critical micelle concentrations at various NaCl concentrations by pyrene fluorescence probe, and the degree of counterion binding to micelle was determined using the Corrin-Harkins plots. The degree of counterion binding was found to be 0.37-0.38 for chenodeoxycholate conjugates, while the determination of the degree was quite difficult for ursodeoxycholate conjugates. The change of I1/I3 values on the fluorescence spectrum with the conjugate bile salt concentration suggested two steps for their bile salt aggregation. The first step is a commencement of smaller aggregates, the first cmc, and the second one is a starting of stable aggregates, the second cmc. The aggregation number was determined at 308.2 K and 0.15 M NaCl concentration by static light scattering: 16.3 and 11.9 for sodium NaGCDC and NaTCDC, and 7.9 and 7.1 for NaGUDC and NaTUDC, respectively. The solubilization of cholesterol into the bile salt micelles in the presence of coexisting cholesterol phase and the maximum additive concentration (MAC) of cholesterol was determined against the bile salt concentration. The standard Gibbs energy change for the solubilization was evaluated, where the micelles were regarded as a chemical species. The solubilization was stabilized in the order of NaGUDC approximately = NaTUDC < NaTC < NaGC < NaTCDC < NaGCDC < NaTDC < NaGDC, where the preceding results were taken into the order.     

Effect of p75NTR on the regulation of naturally occurring cell death and retinal ganglion cell number in the mouse eye

Neurotrophins induce neural cell survival and differentiation during retinal development and regeneration through the high-affinity tyrosine kinase (Trk) receptors. On the other hand, nerve growth factor (NGF) binding to the low-affinity neurotrophin receptor p75 (p75(NTR)) might induce programmed cell death (PCD) in the early phase of retinal development. In the present study, we examined the retinal cell types that experience p75(NTR)-induced PCD and identify them to be postmitotic retinal ganglion cells (RGCs). However, retinal morphology, RGC number, and BrdU-positive cell number in p75(NTR) knockout (KO) mouse were normal after embryonic day 15 (E15). In chick retina, migratory RGCs express p75(NTR), whereas layered RGCs express the high-affinity NGF receptor TrkA, which may switch the pro-apoptotic signaling of p75(NTR) into a neurotrophic one. In contrast to the chick model, migratory RGCs express TrkA, while stratified RGCs express p75(NTR) in mouse retina. However, RGC number in TrkA KO mouse was also normal at birth. We next examined the expression of transforming growth factor beta (TGFbeta) receptor, which modulates chick RGC number in combination with p75(NTR), but was absent in mouse RGCs. p75(NTR) and TrkA seem to be involved in the regulation of mouse RGC number in the early phase of retinal development, but the number may be later adjusted by other molecules. These results suggest the different mechanism of RGC number control between mouse and chick retina.     

Effect of docosahexaenoic acid intake on lipid peroxidation in diabetic rat retina under oxidative stress

Docosahexaenoic acid (DHA) plays an important role in visual function but has a highly oxidation-prone chemical structure. Therefore, we investigated how dietary DHA affects the generation of lipid peroxides in rat retina under oxidative stress in diabetes with/without vitamin E (VE) deficiency. Streptozotocin-induced (50 mg i.p./kg B.W.) diabetic Sprague-Dawley (SD) rats were assigned to four groups: (i) control/VE(+), (ii) DHA/VE(+), (iii) control/VE( - ) and (iv) DHA/VE( - ), and raised for 28 days. We then measured lipid peroxide levels in the retina, serum and liver. With a normal intake of VE, dietary DHA increased only the retinal level of thiobarbituric acid-reactive substances (TBARS) slightly. In contrast, in rats with VE deficiency, dietary DHA increased serum and liver lipid peroxide levels but not in the retina. These results suggest that dietary DHA does not necessarily promote lipid peroxidation in the retina even under high oxidative stress.     

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-γ and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4⁺CD45RO⁺ T cells from peripheral blood, the percentage of ICOS⁺ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [³H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-γ, IL-4 and IL-10 in both SLE and normal T cells. IFN-γ in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.