The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells

Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1^GT/GT) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1^GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation.     

Ketogenic essential amino acids replacement diet ameliorated hepatosteatosis with altering autophagy-associated molecules

Ketogenic amino acid (KAA) replacement diet has been shown to cure hepatic steatosis, a serious liver disease associated with diverse metabolic defects. In this study, we investigated the effects of KAA replacement diet on nutrition sensing signaling pathway and analyzed whether induction of hepatic autophagy was involved. Mice are fed with high fat diet (HFD) or KAA replacement in high-fat diet (30% fat in food; HFD)-fed (HFD^KAAR) and sacrificed at 8, 12, 16 weeks after initiation of experimental food. Hepatic autophagy was analyzed in protein expression of several autophagy-associated molecules and in light chain-3 green fluorescent protein (LC-3 GFP) transgenic mice. HFD^KAAR showed increased AMP-activated protein kinase (AMPK) phosphorylation and enhanced liver kinase B1 (LKB1) expression compared to control HFD-fed mice. The KAA-HFD-induced activation of AMPK was associated with an increased protein expression of sirtuin 1 (Sirt1), decreased forkhead box protein O3a (Foxo3a) level, and suppression of mammalian target of rapamycin (mTOR) phosphorylation compared with the HFD-fed mice. The intervention study revealed that a KAA-replacement diet also ameliorated all the established metabolic and autophagy defects in the HFD-fed mice, suggesting that a KAA-replacement diet can be used therapeutically in established diseases. These results indicate that KAA replacement in food could be a novel strategy to combat hepatic steatosis and metabolic abnormalities likely involvement of an induction of autophagy.     

Studies on the Proteolytic Action of Dairy Lactic Acicl Bacteria

Aseptic rennet curd prepared under the aseptic conditions and Str. cremoris- and L. helveticus-cheese prepared by sandwiching the cell pellets of Str. cremoris and L. helveticus between aseptic rennet curd, respectively, were ripened at 10°C for desired period.

Water soluble nitrogen (WSN) contents of both aseptic rennet curd and two kinds of cheese were determined. Gradual increase of WSN content of aseptic rennet curd was recognized all through the ripening preiod. WSN contents of both Str. cremoris- and L. helveticus-cheese were remarkably higher than those of aseptic rennet curd after 12 days ripening. This tendency was more remarkably recognized after 60 or 70 days ripening. α^s-Casein was mainly hydrolyzed by these lactic acid bacteria during ripening. α^s-Casein in two kinds of the cheese was more easily degradated by these lactic acid bacteria than that in aseptic rennet curd by rennet.

Judging from the results in previous and present reports, it was estimated that lactic acid bacteria used as a starter began to autolyze after 12 days ripening and that intracellular proteases released from their cells mainly hydrolyzed α^s-casein contained in Ca-paracaseinate of aseptic rennet curd to water soluble substances. This hydrolysis was also estimated from the viscous texture observed by scanning electron micrography.     

Contribution of a Salt Bridge Triad to the Thermostability of a Highly Alkaline Protease from an Alkaliphilic Bacillus Strain*

Summary Crystallographic analysis of the highly alkaline M-protease from an alkaliphilic Bacillus strain shows the occurrence of a unique salt bridge triad Arg19–Glu271–Arg275 (in subtilisin BPN′ numbering), which is not found in less alkaline true subtilisins BPN′ and Carlsberg from Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Because the corresponding residues are all Gln residue in the subtilisin BPN′, Gln residue was engineered into the position(s) 19, 271 and/or 275 in M-protease by site-directed mutagenesis. Disruptions of the salt bridge caused the reduction of the thermostability of the mutant proteins at alkaline pH with the following decreasing order of thermal inactivation rate; the wild-type > Arg275 → Gln > Glu271 → Gln > Arg19 → Gln/Glu271 → Gln/Arg275 → Gln > Arg19 → Gln. This result provides the evidence that the salt bridge triad contributes to the thermostability and structural rigidity of the highly alkaline M-protease.     

Molecular design of fluorescent labeled glycosides as acceptor substrates for sialyltransferases

A series of dansyl-labeled glycosides with di-, tetra-, and hexasaccharides carrying the terminal N-acetyllactosamine (LacNAc) sequence were synthesized as acceptor substrates for α2,6- and α2,3-sialyltransferases. As an alternative design, dansyl-labeled LacNAc glycoside carrying a long-spacer linked glycan was engineered by replacement of the LacNAc or lactose units with an alkyl chain. In addition, we designed a dansyl-labeled bi-antennary LacNAc glycoside as an N-linked oligosaccharide mimetic, such as asialo-α(1)-acid glycoprotein. The kinetic parameters for the transfer reaction of synthesized dansyl-labeled glycosides by sialyltransferases were determined by the fluorescent HPLC method. The catalytic efficiencies (V(max)/K(m)) of acceptor substrates carrying the terminal LacNAc sequence with various length glycans in the array for α2,6- and α2,3-sialyltransferases decreased in a glycan length-dependent manner. Furthermore, of the acceptor substrates tested, dansyl-labeled bi-antennary LacNAc glycoside displayed the most favorable K(m) value for α2,6- and α2,3-sialyltransferases.     

pH-dependent equilibrium between long lived near-UV intermediates of photoactive yellow protein

The long lived intermediate (signaling state) of photoactive yellow protein (PYP(M)), which is formed in the photocycle, was characterized at various pHs. PYP(M) at neutral pH was in equilibrium between two spectroscopically distinct states. Absorption maxima of the acidic form (PYP(M)(acid)) and alkaline form (PYP(M)(alkali)) were located at 367 and 356 nm, respectively. Equilibrium was represented by the Henderson-Hasselbalch equation, in which apparent pK(a) was 6.4. Content of alpha- and/or beta-structure of PYP(M)(acid) was significantly greater than PYP(M)(alkali) as demonstrated by the molar ellipticity at 222 nm. In addition, changes in amide I and II modes of beta-structure in the difference Fourier transform infrared spectra for formation of PYP(M)(acid) was smaller than that of PYP(M)(alkali). The vibrational mode at 1747 cm(-1) of protonated Glu-46 was found as a small band for PYP(M)(acid) but not for PYP(M)(alkali), suggesting that Glu-46 remains partially protonated in PYP(M)(acid), whereas it is fully deprotonated in PYP(M)(alkali). Small angle x-ray scattering measurements demonstrated that the radius of gyration of PYP(M)(acid) was 15.7 Angstroms, whereas for PYP(M)(alkali) it was 16.2 Angstroms. These results indicate that PYP(M)(acid) assumes a more ordered and compact structure than PYP(M)(alkali). Binding of citrate shifts this equilibrium toward PYP(M)(alkali). UV-visible absorption spectra and difference infrared spectra of the long lived intermediate formed from E46Q mutant was consistent with those of PYP(M)(acid), indicating that the mutation shifts this equilibrium toward PYP(M)(acid). Alterations in the nature of PYP(M) by pH, citrate, and mutation of Glu-46 are consistently explained by the shift of the equilibrium between PYP(M)(acid) and PYP(M)(alkali).     

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.     

Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single‐molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force‐extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm‐like chain model, the contour lengths calculated from the peaks in the profile suggested that linked‐GroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.     

Oxytocin hypersensitivity in pregnant P-LAP deficient mice

AimsOxytocin (OT) is the strongest uterotonic substance and has been used widely to induce labor. The physiological importance of OT in modulating the initiation and progression of labor remains unclear. In this study, we showed the roles of OT with onset of labor and also the arginine vasopressin (AVP) effect on urine volume in vivo using both wild type (WT) and placental leucine aminopeptidase (P-LAP)-deficient (KO) mice.Main methodsOT (1, 2, 2.5 U/day) or recombinant P-LAP (0.01 U/day) was continuously infused from gestation day 15.5 in WT and P-LAP KO mice. Duration until onset of labor was observed. Before and after administration of AVP (1 U/day) in WT and P-LAP KO mice, urine volume was measured.Key findingsA significant shortening of pregnancy term was observed in P-LAP KO mice. Continuous infusion of OT (1 U/day) revealed that P-LAP KO mice resulted in premature delivery (OT hypersensitivity). We could observe a significant decrease of urine volume in P-LAP KO mice by administration of AVP. Administration of recombinant P-LAP in WT mice resulted in the delay of the onset of labor about 1.5 days compared with control mice.SignificanceOur present study shows that the regulation of the onset of labor mainly depends on OT and its degradation by P-LAP and also the possible role of P-LAP in the regulation of urine output. P-LAP might be involved in the increased OT sensitivity just prior to onset of labor and also in the onset of labor by degradation of OT.     

Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme

Glycoconjugate Journal - Glycogen debranching enzyme (GDE) is bifunctional in that it exhibits both 4-α-glucanotransferase and amylo-α-1,6-glucosidase activity at two distinct catalytic...