Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder. Here, we report a novel mechanism for the occurrence of DMD in females. In a Vietnamese DMD girl, conventional PCR amplification analysis disclosed a deletion of exons 12–19 of the dystrophin gene on Xp21.2, with a karyotype of 46, XY. Furthermore, a novel mutation in the androgen-receptor gene on Xq11.2-q12 was identified in this girl, which led to male pseudohermaphroditism. Co-occurrence of mutations of these two genes constitutes a novel mechanism underlying female DMD.     

Effects of chloromethanes on growth of and deletion of the pce gene cluster in dehalorespiring Desulfitobacterium hafniense strain Y51

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 microM, but not carbon dichloride even at 1 mM.     

Quantitative determination of callose in tree roots

The formation of callose in tree roots has been suggested as a physiological indicator of aluminum (Al) toxicity. Quantifying callose in the roots in forest soils, however, is hampered by the presence of autofluorescent materials in the roots that disturb the measurement of callose by fluorescence spectrophotometry. Tannins in the roots cause these measurement problems. Here we report on the measurement of callose in the root apices of European chestnut (Castanea sativa) seedlings collected in an acidified forest soil. The callose was quantified with a modified protocol which included three washing steps with polyvinylpolypyrrolidone (PVPP) before the callose was extracted. This procedure reduced the autofluorescence by about 50%. With the use of water or ethanol alone, callose could be measured in only about 15% of the root samples, whereas with the use of PVPP callose could be determined in 95% of the samples. This improved method could help to evaluate the effects of Al toxicity on tree roots grown in forest soils, where callose is detected as a physiological indicator.     

Identification and characterization of a neutralizing monoclonal antibody for the epitope on HM-1 killer toxin

Killer toxin-neutralizing monoclonal antibody (nmAb-KT) against HM-1 killer toxin (HM-1) produced by yeast Williopsis saturnus var. mrakii IFO 0895 reduces both the killing and glucan synthase inhibitory activity of HM-1. nmAb-KT is classified as IgG1kappa and has been shown to be ineffective against HYI killer toxin produced by the related yeast W. saturnus var. saturnus IFO 0117. To determine the epitope for nmAb-KT, overlapping peptides were synthesized from the primary structure of HM-1. nmAb-KT reacted with peptides P5 (33NVHWMVTGGST43), P6 (39TGGSTDGKQG48) and P7 (44DGKQGCATIWEGS56), which represent the middle region of the HM-1 sequence. P6 reacted most strongly with nmAb-KT. Combined analysis by immunoblotting, surface plasmon resonance (SPR) analysis and yeast growth inhibition assay showed that nmAb-KT recognizes a specific epitope within peptide P6. The K(d) value of nmAb-KT against HM-1 and P6 were determined to be 5.48 x 10(-9) M and 1.47 x 10(-6) M by SPR analysis, respectively. These results strongly indicate that nmAb-KT binds to HM-1 at the sequence 41GSTDGK46, and not to HYI at the same position. The potential active site of HM-1 involved in the killing activity against sensitive yeast is discussed.     

A novel phosphatidylcholine which contains pentadecanoic acid at sn-1 and docosahexaenoic acid at sn-2 in Schizochytrium sp. F26-b

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."     

The 1.48 A resolution crystal structure of the homotetrameric cytidine deaminase from mouse

Cytidine deaminase (CDA) is a zinc-dependent enzyme that catalyzes the deamination of cytidine or deoxycytidine to form uridine or deoxyuridine. Here we present the crystal structure of mouse CDA (MmCDA), complexed with either tetrahydrouridine (THU), 3-deazauridine (DAU), or cytidine. In the MmCDA-DAU complex, it clearly demonstrates that cytidine is distinguished from uridine by its 4-NH(2) group that acts as a hydrogen bond donor. In the MmCDA-cytidine complex, cytidine, unexpectedly, binds as the substrate instead of the deaminated product in three of the four subunits, and in the remaining subunit it binds as the product uridine. Furthermore, the charge-neutralizing Arg68 of MmCDA has also exhibited two alternate conformations, I and II. In conformation I, the only conformation observed in the other structurally known homotetrameric CDAs, Arg68 hydrogen bonds Cys65 and Cys102 to modulate part of their negative charges. However, in conformation II the side chain of Arg68 rotates about 130 degrees around the Cgamma-Cdelta bond and abolishes these hydrogen bonds. The lack of hydrogen bonding may indirectly weaken the zinc-product interaction by increased electron donation from cysteine to the zinc ion, suggesting a novel product-expelling mechanism. On the basis of known structures, structural analysis further reveals two subclasses of homotetrameric CDAs that can be identified according to the position of the charge-neutralizing arginine residue. Implications for CDA-RNA interaction have also been considered.     

Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy

The Fc portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C(H)2 domain of each heavy chain with microheterogeneities depending on physiological and pathological states. These N-glycans are known to be essential for promotion of proper effector functions of IgG such as complement activation and Fcgamma receptor (FcgammaR)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgG1-Fc and analyzed their interactions with human soluble FcgammaRIIIa (sFcgammaRIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods. Progressive but less pronounced reductions in the affinity for sFcgammaRIIIa were observed as a result of the galactosidase and subsequent N-acetylhexosaminidase treatments of IgG1-Fc. The following endoglycosidase D treatment, giving rise to a disaccharide structure composed of a fucosylated GlcNAc, abrogated the affinity of IgG1-Fc for sFcgammaRIIIa. On the other hand, those glycosidase treatments did not significantly affect the affinity of IgG1-Fc for protein A. Inspection of stable-isotope-assisted NMR data of a series of Fc glycoforms indicates that the stepwise trimming out of the carbohydrate residues results in concomitant increase in the number of amino acid residues perturbed thereby in the C(H)2 domains. Furthermore, the cleavage at the GlcNAcbeta1-4GlcNAc glycosidic linkage induced the conformational alterations of part of the lower hinge region, which makes no direct contact with the carbohydrate moieties and forms the major FcgammaR-binding site, while the conformation of the C(H)2/C(H)3 interface was barely perturbed that is the protein A-binding site. These results indicate that the carbohydrate moieties are required for maintaining the structural integrity of the FcgammaR-binding site.     

Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor beta in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal

Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor beta (PDGFR-beta) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-beta and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-beta and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-beta and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-beta and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.     

Phosphatidylglucoside exists as a single molecular species with saturated fatty acyl chains in developing astroglial membranes

We previously found that phosphatidylglucoside (PtdGlc), a novel glycolipid expressed in HL60 cells, plays a role in forming signaling microdomains involved in cellular differentiation. Because cells contain minute levels of PtdGlc, pure PtdGlc is very difficult to isolate. Thus, its complete structure has never been assessed. To aid in analyzing PtdGlc, we generated a PtdGlc-specific monoclonal antibody, DIM21, by immunizing mice with detergent-insoluble membranes isolated from HL60 cells [Yamazaki, Y., et al. (2006) J. Immunol. Methods 311, 106-116]. DIM21 immunostaining of murine CNS tissues revealed stage- and cell type-specific localization of the DIM21 antigen during development, with especially high levels of expression in radial glia/astroglia. DIM21 immunostained cultured hippocampal astroglia in a punctate fashion. To characterize the structure of PtdGlc, we isolated DIM21 antigen from fetal brains. Using successive column chromatography, we purified two previously unrecognized glycolipids, PGX-1 and PGX-2, from embryonic day 21 rat brains. DIM21 reacted more strongly to PGX-2 than to PGX-1. Structural analyses with 600 MHz (1)H NMR, FT-ICR mass spectrometry, and GC revealed that PGX-1 is phosphatidyl beta-d-(6-O-acetyl)glucopyranoside and PGX-2 is phosphatidyl beta-d-glucopyranoside. The yields of PGX-1 and PGX-2 were approximately 250 +/- 150 and 440 +/- 270 nmol/g of dried brains, respectively. Surprisingly, both glycolipids were composed exclusively of C18:0 at the C1 position and C20:0 at the C2 position of the glycerol backbone. This saturated fatty acyl chain composition comprising a single molecular species rarely occurs in known mammalian lipids and provides a molecular basis for why PtdGlc resides in raftlike lipid microdomains.