Synthesis of 2-[(2-pyridyl)amino]ethyl beta-D-lactosaminide and evaluation of its acceptor ability for sialyltransferase: a comparison with 4-methylumbelliferyl and dansyl beta-D-lactosaminide

We reported the synthesis of beta-D-lactosaminide with a 2-aminopyridyl group that is linked to a glycosyl tether at the reducing end. This fluorescent disaccharide acts as an acceptor for both alpha-(2-->6)- and alpha-(2-->3)-sialyltransferases. In addition, the acceptor ability of this disaccharide was evaluated and compared with that of beta-D-lactosaminide having a dansyl or a 4-methylumbelliferyl group.     

Cloning of the Ruminococcus albus cel5D and cel9A genes encoding dockerin module-containing endoglucanases and expression of cel5D in Escherichia coli

An EcoRI chromosomal DNA fragment of Ruminococcus albus F-40 that conferred endoglucanase activity on Escherichia coli was cloned. An open reading frame (ORF1) and another incomplete reading frame (ORF2) were found in the EcoRI fragment. The ORF2 was completed using inverse PCR genome walking technique. ORF1 and ORF2, which confront each other, encoded cellulases belonging to families 5 and 9 of the glycoside hydrolases and were designated cel5D and cel9A respectively. The cel5D gene encodes 753 amino acids with a deduced molecular weight of 83,409. Cel5D consists of a signal peptide of 24 amino acids, a family-5 catalytic module, a dockerin module, and two family-4 carbohydrate-binding modules (CBMs). The cel9A gene encodes 936 amino acids with a deduced molecular weight of 104,174, consisting of a signal peptide, a family-9 catalytic module, a family-3 CBM, and a dockerin module. The catalytic module polypeptide (rCel5DCat) derived from Cel5D was constructed, expressed, and purified from a recombinant E. coli. The truncated enzyme hydrolyzed cellohexaose, cellopentaose, and cellotetraose to yield mainly cellotriose and cellobiose with glucose as a minor product, but the enzyme was less active toward cellotriose and not active toward cellobiose, suggesting that this enzyme is a typical endoglucanase. rCel5DCat had a Km of 3.9 mg/ml and a Vmax of 37.2 micromol/min/mg for carboxymethycellulose.     

O2 consumption of mechanically unloaded contractions of mouse left ventricular myocardial slices

Left ventricular (LV) myocardial slices were isolated from murine hearts (300 microm thick) and were stimulated at 1 Hz without external load. Mean myocardial slice O(2) consumption (MVo(2)) per minute (mMVo(2)) without stimulation was 0.97 +/- 0.14 ml O(2).min(-1).100 g LV(-1) and mean mMVo(2) with stimulation increased to 1.80 +/- 0.17 ml O(2).min(-1).100 g LV(-1) in normal Tyrode solution. Mean DeltamVo(2) (the mMVo(2) with stimulation - the mMVo(2) without stimulation) was 0.83 +/- 0.12 ml O(2).min(-1).100 g LV(-1). There were no differences between mean mMVo(2) with and without stimulation in Ca(2+)-free solution. The increases in extracellular Ca(2+) concentrations up to 14.4 mM did not affect the mMVo(2) without stimulation but significantly increased the mMVo(2) with stimulation up to 140% of control. The DeltamMVo(2) significantly increased up to 190% of the control in a dose-dependent manner. In contrast, the shortening did not increase in a dose-dependent manner. Cyclopiazonic acid (CPA; 30 microM) significantly reduced the DeltamMVo(2) to 0.27 +/- 0.06 ml O(2).min(-1).100 g LV(-1) (35% of control). The combination of 5 mM 2,3-butanedione monoxime (BDM) and 30 microM CPA did not further decrease DeltamMVo(2). Although BDM (3-5 mM) decreased the DeltamMVo(2) by 28-30% of control in a dose-independent manner, 3-5 mM BDM decreased shortening in a dose-dependent manner. Our results indicate that the DeltamMVo(2) of mouse LV slices during shortening under mechanically unloaded conditions consists of energy expenditure for total Ca(2+) handling during excitation-contraction coupling, basal metabolism, but no residual cross-bridge cycling.     

Reversible effects of isoproterenol-induced hypertrophy on in situ left ventricular function in rat hearts

The aim of the present study was to evaluate specifically left ventricular (LV) function in rat hearts as they transition from the normal to hypertrophic state and back to normal. Either isoproterenol (1.2 and 2.4 mg.kg(-1).day(-1) for 3 days; Iso group) or vehicle (saline 24 microl.day(-1) for 3 days; Sa group) was infused by subcutaneous implantation of an osmotic minipump. After verifying the development of cardiac hypertrophy, we recorded continuous LV pressure-volume (P-V) loops of in situ ejecting hypertrophied rat hearts. The curved LV end-systolic P-V relation (ESPVR) and systolic P-V area (PVA) were obtained from a series of LV P-V loops in the Sa and Iso groups 1 h or 2 days after the removal of the osmotic minipump. PVA at midrange LV volume (PVA(mLVV)) was taken as a good index for LV work capability (13, 15, 20, 21). However, in rat hearts during remodeling, whether PVA(mLVV) is a good index for LV work capability has not been determined yet. In the present study, in contrast to unchanged end-systolic pressure at midrange LV volume, PVA(mLVV) was significantly decreased by isoproterenol treatment relative to saline; however, these measurements were the same 2 days after pump removal. Simultaneous treatment with a beta(1)-blocker, metoprolol (24 mg.kg(-1).day(-1)), blocked the formation of cardiac hypertrophy and thus PVA(mLVV) did not decrease. The reversible changes in PVA(mLVV) reflect precisely the changes in LV work capability in isoproterenol-induced hypertrophied rat hearts mediated by beta(1)-receptors. These results indicate that the present approach may be an appropriate strategy for evaluating the effects of antihypertrophic and antifibrotic modalities.     

Spermidine synthase genes are essential for survival of Arabidopsis

The cellular polyamines putrescine, spermidine, and spermine are ubiquitous in nature and have been implicated in a wide range of growth and developmental processes. There is little information, however, on mutant plants or animals defective in the synthesis of polyamines. The Arabidopsis genome has two genes encoding spermidine synthase, SPDS1 and SPDS2. In this paper, we describe T-DNA insertion mutants of both of these genes. While each mutant allele shows normal growth, spds1-1 spds2-1 double-mutant seeds are abnormally shrunken and they have embryos that are arrested morphologically at the heart-torpedo transition stage. These seeds contain significantly reduced levels of spermidine and high levels of its precursor, putrescine. The embryo lethal phenotype of spds1-1 spds2-1 is complemented by the wild-type SPDS1 gene. In addition, we observed a nearly identical seed phenotype among an F2 seed population from the cross between the spds2-1 allele and SPDS1 RNA interference transgenic lines. These data provide the first genetic evidence indicating a critical role of the spermidine synthase in plant embryo development.     

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.     

PACSIN3 binds ADAM12/meltrin alpha and up-regulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor

A disintegrin and metalloprotease 12 (ADAM12/meltrin alpha) is a key enzyme implicated in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor (proHB-EGF)-dependent epidermal growth factor receptor (EGFR) transactivation. However, the activation mechanisms of ADAM12 are obscure. To determine how ADAM12 is activated, we screened proteins that bind to the cytoplasmic domain of ADAM12 using a yeast two-hybrid system and identified a protein called PACSIN3 that contains a Src homology 3 domain. An analysis of interactions between ADAM12 and PACSIN3 using glutathione S-transferase fusion protein revealed that a proline-rich region (amino acid residues 829-840) of ADAM12 was required to bind PACSIN3. Furthermore, co-immunoprecipitation and co-localization analyses of ADAM12 and PACSIN3 proteins also revealed their interaction in mammalian cells expressing both of them. The overexpression of PACSIN3 in HT1080 cells enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced proHB-EGF shedding. Furthermore, knockdown of endogenous PACSIN3 by small interfering RNA in HT1080 cells significantly attenuated the shedding of proHB-EGF induced by TPA and angiotensin II. Our data indicate that PACSIN3 has a novel function as an up-regulator in the signaling of proHB-EGF shedding induced by TPA and angiotensin II.     

Essential role of Fc gamma receptors in anti-type II collagen antibody-induced arthritis

Anti-type II collagen (anti-CII) Ab is a well-known autoantibody observed in patients with rheumatoid arthritis. Injection of anti-CII Ab and LPS induces arthritis in mice in which anti-CII Ab as well as inflammatory cytokines, IL-1beta and TNF-alpha, play critical roles. We investigated the involvement of IgG FcRs (FcgammaRs) in this arthritis model. BALB/c mice injected with the F(ab')(2) of anti-CII Ab showed no signs of arthritis. Arthritis development was not observed in FcRgamma(-/-) mice and was partially suppressed in FcgammaRIII(-/-) mice despite the binding of anti-CII Ab and C3 to cartilage surface. Surprisingly, BALB/c mice lacking FcgammaRIIB, which is known as an inhibitory FcgammaR, developed arthritis with no exacerbation in arthritis score compared with wild-type (WT) mice, and only slight exacerbation was observed in the histopathological analysis. In contrast, aged FcgammaRIIB(-/-) BALB/c mice developed arthritis without LPS injection, suggesting an augmented susceptibility to arthritis in aged FcgammaRIIB(-/-) mice. No significant difference was observed among BALB/c-WT, -FcRgamma(-/-), and -FcgammaRIIB(-/-) mice on cytokine production induced by anti-CII Ab and LPS injection. Severe arthritis developed in BALB/c-WT and -FcgammaRIIB(-/-) mice, but not in BALB/c-FcRgamma(-/-) mice, after the injection of anti-CII Ab and inflammatory cytokines. These results suggest that the reason behind the nondevelopment of arthritis in FcRgamma(-/-) BALB/c mice is not due to a disorder in transient cytokine production, but to an irregularity downstream of cytokine production.     

Synthesis and analysis of nucleosides bearing pyrrolepolyamide binding to DNA

An efficient synthesis of adenosine bearing pyrrolepolyamide 1 was achieved by coupling of 3 with 2. The CD spectra obtained at several [ligand ]/[duplex] ratios allowed verification of the formation complex of the DNA duplex [d(CGCAAATTGGC)/d(GCCAATTTGCG)] with 1.