Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.     

Neutrophils as a source of cytokines in inflammation

The recruitment of neutrophils into inflammatory foci is a fundamental process observed in inflammation. The function of neutrophils has long been regarded only as an effector cell that kills the invading pathogens. Recent evidence has demonstrated that neutrophils are capable of producing inflammatory cytokines. The findings are, however, mainly based on the findings obtained in vitro. It has not been fully elucidated if neutrophils could synthesize and secrete cytokines in vivo. Animal models of inflammation are essential to address the issue and provide insight into the involvement of neutrophils in producing cytokines.     

Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose

Lacto-N-tetraose (Galbeta1 -3GlcNAcbeta1-3Galbeta1-4Glc, LNT) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, LNnT) were enzymatically synthesized by consecutive additions of GlcNAc and Gal residues to lactose. Lacto-N-triose II (GlcNAcbeta1-3Galbeta1-4Glc) was prepared first by the transfer of GlcNAc from UDP-GlcNAc to lactose by beta-1,3-N-acetylglucosaminyltransferase from bovine serum. The resulting lacto-N-triose II was converted into LNT and LNnT utilizing two kinds of beta-D-galactosidase-mediated transglycosylations. Thus, beta-D-galactosidase from Bacillus circulans ATCC31382 induced regioselective galactosyl transfer from o-nitrophenyl beta-D-galactoside to the OH-3" position of lacto-N-triose II, and commercially available beta-D-galactosidase from B. circulans to the OH-4" position of lacto-N-triose II. These convenient processes are suitable for large-scale preparations of LNT and LNnT. As another method, LNT was directly synthesized from lactose as an initial substance, utilizing lacto-N-biosidase (Aureobacterium sp. L-101)-mediated transglycosylation with Galbeta1-3GlcNAcbeta-pNP donor.     

Increased numbers of B-1 cells and enhanced responses against TI-2 antigen in mice lacking APS, an adaptor molecule containing PH and SH2 domains

APS (adaptor molecule containing PH and SH2 domains) is an intracellular adaptor protein that forms an adaptor family along with Lnk and SH2-B. While experiments using cultured cell lines have demonstrated that APS is phosphorylated in response to various stimuli, its in vivo functions remain unclear. We attempted to determine the physiological roles of APS by generating APS-deficient (APS(-/-)) mice. APS(-/-) mice were viable and fertile and showed no abnormalities or growth retardation. Immunologically, APS(-/-) mice showed normal development and distribution of lymphocytes and myeloid cells, except for increased numbers of B-1 cells in the peritoneal cavity. APS(-/-) mice exhibited an enhanced humoral immune response against trinitrophenol-Ficoll, a thymus-independent type 2 antigen, while APS(-/-) B-2 cells exhibited normal proliferative responses and tyrosine phosphorylation of intracellular proteins upon B-cell receptor (BCR) cross-linking. APS colocalized with filamentous actin (F-actin) accumulated during the capping of BCRs in APS-transgenic B cells. After BCR stimulation, F-actin contents were lower in APS(-/-) B-1 cells than in wild-type B-1 cells. Our results indicate that APS might have a novel regulatory role in actin reorganization and control of B-1 cell compartment size.     

Chloroplast phylogeny indicates that bryophytes are monophyletic

Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other data sets. We propose bryophyte monophyly as the currently best hypothesis.     

Development of damage function of acidification for terrestrial ecosystems based on the effect of aluminum toxicity on net primary production

Background  Acidification is one of the important impact categories for life cycle impact assessment. Although its characterization has progressed during this decade through the employment of midpoint approaches, only limited studies of endpoint approaches have been performed. Objective. This study aimed at developing damage function of acidification for terrestrial ecosystems in Japan. Damage function expresses a quantitative relationship between the inventory and endpoint damage. Methods  The geographical boundary was limited in Japan both for emission and impact. In this study, sulfur dioxide (SO₂), nitrogen monoxide (NO), nitrogen dioxide (NO₂) (NO and NO₂ collectively mean NO_x), hydrogen chloride (HC1), and ammonia (NH₃) were considered as major causative substances of acidification. Net primary production (NPP) of existing vegetation was adopted as an impact indicator of terrestrial ecosystems. The aluminum toxicity was adopted as the major factor of effect on terrestrial ecosystems due to acidification. The leachate concentration of monomeric inorganic aluminum ions was selected to express the plant toxicity of aluminum. Results and Discussion  The results of damage function gave utilizable factors both for a midpoint approach and an endpoint approach; Atmospheric Deposition Factor (ADF) and Damage Factor (DF) applicable to the former and the latter, respectively. The ADF indicates an increase of H⁺ deposition per unit area to an additional emission of causative sustance. The additional emission corresponds to some alternatives in industry, not the baseline emission. The DF indicates the total NPP damage in all of Japan due to the additional emission of causative substances. The derived NPP damage is on the order of one millionth of the NPP itself. HC1 and NH3 showed larger ADFs and DFs than that of SO₂ and NO_x. The reason was ascribed to the relatively large source-receptor relationships (SRR) of HC1 and NH₃. However, since the method applied to determine the SRR of HC1 and NH₃ has larger uncertainties than that of SO₂ and NO_x, attention is needed to handle the difference. Conclusion  The damage function easily defines the concrete NPP damage due to an additional emission. The impact indica tor, NPP, also has an advantage in its mass unit that is directly summable through the entire impact categories. Expansion of endpoints, such as in aquatic ecosystems, material degradation, human health, and biodiversity aspects of terrestrial ecosystems, is an important subject for future work. Further, uncertain analyses for major parameters will provide helpful information on the reliability of damage function.     

Role of follicular dendritic cells in the early HIV-1 infection: in vitro model without specific antibody

About 90% of HIV-1 RNA in the lymph nodes is reported to localize in follicular dendritic cellsnetwork (FDC-NW) as early as several days after infection and as much as that in the late stage. But the mechanism remains to be fully understood. To elucidate the role of follicular dendritic cells (FDC) in the early stage of HIV-1 infection, FDC-like cell strains (FDCLC) were established and they were characterized in the co-culture system with T cells for their effect on HIV-1 trapping and replication in p24 immunoassay, immunohistochemistry as well as confocal and electronmicroscopy. Established FDCLC were positive for CNA-42, S-100alpha and intercellular desmosome-like junctions. L-SIGN and DC-SIGN were also detected in FDCLC. Alu-HIV-1 PCR analysis showed no HIV-1 integration in FDCLC. FDCLC trapped HIV-1 and transferred them to uninfected MOLT-4 T cells (MOLT-4) efficiently in the absence of specific antibody. FDCLC also accelerated HIV-1 replication in HIV-1-pre-exposed MOLT-4. These unique FDCLC effects were explained, at least partly, by the fact that FDCLC up-regulated CD4 expression in MOLT-4 and helped T cells escape from apoptosis in the co-culture. These data suggest that FDC/FDCLC engage not only in trapping but also in active expansion of HIV-1 in the absence of specific antibody.     

New analogue of arenastatin A, a potent cytotoxic spongean depsipeptide, with anti-tumor activity

Two analogues possessing steric hindered substituents on C-15 of arenastatin A (1), a potent cytotoxic spongean depsipeptide, were synthesized and shown to enhance stability in mouse serum. Notably, 15-tert-butylanalogue (6) with higher cytotoxicity exhibited in vivo anti-tumor activity through iv administration different from 1. Additionally, conformation analysis among the two analogues and arenastatin A (1) indicated that the torsion angle from C-14 to C-20 is a conclusive factor for the potent cytotoxicity of 1.     

Synthesis of anilino-monoindolylmaleimides as potent and selective PKCbeta inhibitors

We report herein synthesis of PKCbeta-selective inhibitors possessing the novel pharmacophore of anilino-monoindolylmaleimide. Several compounds of this series exhibited IC50's as low as 50 nM against human PKCbeta2. One of the most potent compounds, 6l, inhibited PKCbeta1 and PKCbeta2 with IC50 of 21 and 5 nM, respectively, and exhibited selectivity of more than 60-fold in favor of PKCbeta2 relative to other PKC isozymes (PKCalpha, PKCgamma, and PKCepsilon).