Measurement and characterization of C-3 epimerization activity toward vitamin D3

Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.     

Direct determination of a membrane-peptide interface using the nuclear magnetic resonance cross-saturation method

Membrane-peptide interactions are involved in many crucial biological and pharmacological activities. To clarify the interaction mode of membrane-peptide complexes, it is important to analyze both the dynamic properties and the contact residues of the membrane-bound peptide. In this study, we investigated the dynamic properties of a peptide bound to a lipid bilayer, using relaxation and amide-water exchange analyses, and directly determined the membrane-peptide interface, using the cross-saturation method. For the models of a lipid bilayer and a peptide, isotropic bicelles and mastoparan were used, respectively. The results indicate that mastoparan had a heterogeneous distribution of motion over various timescales and interacted with the lipid bilayer by using its hydrophobic side; the molecule was located within the lipid bilayer rather than on the surface, as thought previously. This study shows that the cross-saturation method is useful for determining the interface of not only protein-protein but also membrane-peptide complexes.     

Insertion/deletion polymorphism in clusterin gene influences serum lipid levels and carotid intima-media thickness in hypertensive Japanese females

Clusterin has been implicated in lipid metabolism and atherogenesis, however, the influence of genetic variation has not been examined in Japanese. In this study, we identified 11 single nucleotide polymorphisms (SNPs) of clusterin gene by direct sequencing. Among them, one promoter SNP (-4453T>G), one missense SNP (4183G>A), and 2 common SNPs (5608T>C and 6316delT) were genotyped in 525 asymptomatic hypertensives not treated with lipid lowering agents. -4453T>G, 4183G>A, and 5608T>C showed no correlation with the clinical characteristics, however, in the 6316delT, an insertion (I)/deletion (D) polymorphism, D/D subjects had significantly higher levels of total cholesterol and low-density lipoprotein (LDL)-cholesterol than I/I subjects in females but not in males. Female subjects with the D allele (D/D+I/D) had greater intima-media thickness of the carotid artery than I/I subjects. In a multiple logistic regression analysis, the D allele of 6316delT was detected as an independent predictor for the plaque prevalence. In conclusion, the clusterin gene polymorphism may contribute to the serum lipid levels and the progression of carotid atherosclerosis in hypertensive Japanese females.     

Effect of solar near ultraviolet radiation on growth of Chlorella cells

Cells of Chlorella regularis (Artari) Oltmanns (S-50) were grown under solar radiation in Tokyo, using a newly constructed outdoor culture system. The maximum specific growth rate (log₂ unit h⁻¹) was about 0.3, which was a little lower than the highest value reported using artificial light in the laboratory. The near ultraviolet light, consisting mostly of UV-A, inhibited the growth from 10 to 40% in summer mornings; the inhibition was not significant in the afternoon. The percentage inhibition was correlated with the total dose of the ultraviolet radiation.     

Green fluorescent protein causes mitochondria to aggregate in the presence of the Bcl-2 family proteins

Green fluorescent protein (GFP) has been widely used in a variety of experiments in cell biology. When cells were co-transfected with the GFP gene and the bcl-2 family genes bcl-2, bcl-x(L), and bax, mitochondria appeared to aggregate at the periphery of the nucleus specifically where GFP was expressed. Little aggregation was seen in the presence of other members of the GFP family, EGFP (enhanced GFP), ECFP (enhanced cyan variant), and EYFP (enhanced yellow-green variant). GFP but not EGFP seemed to promote cell death induced by pro-apoptotic Bax. Thus, GFP specifically promotes the aggregation of mitochondria when co-expressed with a member of the Bcl-2 family in association with apoptosis.     

Direct measurement of Ca2+ concentration in the SR of living cardiac myocytes

Although abnormal sarcoplasmic reticulum (SR) Ca(2+) handling may cause heart failure, there has been no method to directly measure Ca(2+) concentration in SR ([Ca(2+)](SR)) of living cardiomyocytes. We have measured [Ca(2+)](SR) by expressing novel fluorescent Ca(2+) indicators yellow cameleon (YC) 2.1, YC3er, and YC4er in cultured neonatal rat cardiomyocytes. The distribution of YC2.1 was uniform in the cytoplasm, while that of YC3er/YC4er, containing the signal sequence which recruits them to SR, showed reticular pattern and was co-localized with SERCA2a. The treatment with caffeine reversibly decreased the emission ratio (R) in YC3er/YC4er-expressing myocytes, and the treatment with ryanodine and thapsigargin decreased R irreversibly. During the contraction-relaxation cycle, R was changed periodically in the YC2.1- and YC3er-expressing myocytes, but its direction of the change was opposite. These results suggest that YC3er/YC4er were specifically localized and functioned in SR as a [Ca(2+)](SR) indicator. This technique would be useful to understand the function of SR in failing myocardium.     

Metabolism of vitamin D by human microsomal CYP2R1

The activation of vitamin D requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney. However, it remains unclear which enzyme is relevant to vitamin D 25-hydroxylation. Recently, human CYP2R1 has been reported to be a potential candidate for a hepatic vitamin D 25-hydroxylase. Thus, vitamin D metabolism by CYP2R1 was compared with human mitochondrial CYP27A1, which used to be considered a physiologically important vitamin D(3) 25-hydroxylase. A clear difference was observed between CYP2R1 and CYP27A1 in the metabolism of vitamin D(2). CYP2R1 hydroxylated vitamin D(2) at the C-25 position while CYP27A1 hydroxylated it at positions C-24 and C-27. The K(m) and k(cat) values for the CYP2R1-dependent 25-hydroxylation activity toward vitamin D(3) were 0.45microM and 0.97min(-1), respectively. The k(cat)/K(m) value of CYP2R1 was 26-fold higher than that of CYP27A1. These results strongly suggest that CYP2R1 plays a physiologically important role in the vitamin D 25-hydroxylation in humans.     

Solution structure and functional characterization of SGTx1, a modifier of Kv2.1 channel gating

SGTx1 is a peptide toxin isolated from the venom of the spider Scodra griseipes that has been shown to inhibit outward K(+) currents in rat cerebellar granule neurons. Although its amino acid sequence is known to be highly (76%) homologous with that of hanatoxin (HaTx), a well-characterized modifier of Kv2.1 channel gating, the structural and functional characteristics of SGTx1 remain largely unknown. Here we describe the NMR solution structure of SGTx1, the mechanism of its interaction with Kv2.1 channels, and its effect on channel activity once bound. The NMR structure of SGTx1 contains a molecular fold closely resembling the "inhibitor cystine knot" of HaTx, which is composed of an antiparallel beta-sheet and four chain reversals stabilized by three disulfide bonds. Functionally, SGTx1 reversibly inhibited K(+) currents in oocytes expressing Kv2.1 channels. Moreover, generation of steady-state activation curves showed that, consistent with other gating modifiers, SGTx1 acted by shifting the activation of the channel to more depolarized voltages. Thus, the surface profile and mechanism of action of SGTx1 are similar to those of HaTx. Still, detailed comparison of SGTx1 with HaTx revealed differences in binding affinity and conformational homogeneity that result from differences in the charge distribution at the binding surface and in the amino acid composition of the respective beta-hairpin structures in the peptides.     

Mucor hiemalis endo-beta-N-acetylglucosaminidase can transglycosylate a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide

We found that the recombinant endo-beta-N-acetylglucosaminidase of Mucor hiemalis (Endo-M) expressed in Candida boidinii had the transglycosylation activity of transferring a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide to the acceptor (p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside) in a good yield of 43%.     

The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process

Several lines of in vitro evidence suggest the potential role of IFN-gamma in angiogenesis and collagen deposition, two crucial steps in the wound healing process. In this report, we examined the role of IFN-gamma in the skin wound healing process utilizing WT and IFN-gamma KO mice. In WT mice, excisional wounding induced IFN-gamma mRNA and protein expression by infiltrating macrophages and T cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-gamma KO mice exhibited an accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-gamma KO mice exhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, compared with WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-gamma KO mice also exhibited accelerated collagen deposition with enhanced production of TGF-beta1 protein in wound sites, compared with WT mice. Furthermore, the absence of IFN-gamma augmented the TGF-beta1-mediated signaling pathway, as evidenced by increases in the levels of total and phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalk between the IFN-gamma/Stat1 and TGF-beta1/Smad signaling pathways in the wound healing process.