Investigations of the transfructosylation reaction by fructosyltransferase from B. subtilis NCIMB 11871 for the synthesis of the sucrose analogue galactosyl-fructoside

The exo-fructosyltransferase produced from B. subtilis NCIMB 11871 strain transfers the fructose moiety from donor alpha12 linked saccharides such as sucrose, raffinose and stachyose to the acceptor d-galactose, leading to the sucrose analogue, galactosyl-fructoside. Here, we report detailed kinetic studies. The enzyme showed a remarkably high optimal temperature at 50 degrees C and was effectively immobilised on Eupergit C 250 L and Trisopor-Amino. This is also the first report about the equilibrium of the transfructosylation reaction, its activation energy determination, the structure of the product and its preparative scale isolation.     

Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates

Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the deltaSH virus was only marginally less efficient than that of wild-type HMPV, whereas the deltaG and deltaM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The deltaG and deltaM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.     

Tissue- and gene-specific recruitment of steroid receptor coactivator-3 by thyroid hormone receptor during development

Numerous coactivators that bind nuclear hormone receptors have been isolated and characterized in vitro. Relatively few studies have addressed the developmental roles of these cofactors in vivo. By using the total dependence of amphibian metamorphosis on thyroid hormone (T3) as a model, we have investigated the role of steroid receptor coactivator 3 (SRC3) in gene activation by thyroid hormone receptor (TR) in vivo. First, expression analysis showed that SRC3 was expressed in all tadpole organs analyzed. In addition, during natural as well as T3-induced metamorphosis, SRC3 was up-regulated in both the tail and intestine, two organs that undergo extensive transformations during metamorphosis and the focus of the current study. We then performed chromatin immunoprecipitation assays to investigate whether SRC3 is recruited to endogenous T3 target genes in vivo in developing tadpoles. Surprisingly, we found that SRC3 was recruited in a gene- and tissue-dependent manner to target genes by TR, both upon T3 treatment of premetamorphic tadpoles and during natural metamorphosis. In particular, in the tail, SRC3 was not recruited in a T3-dependent manner to the target TRbetaA promoter, suggesting either no recruitment or constitutive association. Finally, by using transgenic tadpoles expressing a dominant negative SRC3 (F-dnSRC3), we demonstrated that F-dnSRC3 was recruited in a T3-dependent manner in both the intestine and tail, blocking the recruitment of endogenous coactivators and histone acetylation. These results suggest that SRC3 is utilized in a gene- and tissue-specific manner by TR during development.     

ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion--Part II: shoulder, elbow, wrist and hand

In this communication, the Standardization and Terminology Committee (STC) of the International Society of Biomechanics proposes a definition of a joint coordinate system (JCS) for the shoulder, elbow, wrist, and hand. For each joint, a standard for the local axis system in each articulating segment or bone is generated. These axes then standardize the JCS. The STC is publishing these recommendations so as to encourage their use, to stimulate feedback and discussion, and to facilitate further revisions. Adopting these standards will lead to better communication among researchers and clinicians.     

The oncogenic serine/threonine kinase Pim-1 phosphorylates and inhibits the activity of Cdc25C-associated kinase 1 (C-TAK1): a novel role for Pim-1 at the G2/M cell cycle checkpoint

The Pim-1 oncogene encodes a serine-threonine kinase that relays signals from cytokine receptors and contributes to the formation of lymphoid tumors when expressed at high levels. Here we show that the protein kinase Cdc25 C-associated kinase 1 (C-TAK1) is a binding partner and a substrate of Pim-1. A physical interaction of Pim-1 and C-TAK1 could be shown biochemically and in yeast two-hybrid assays. Immunofluorescence experiments suggested that Pim-1.C-TAK1 complexes are predominantly cytoplasmic. When transiently transfected, Pim-1 was also found in the nucleus and could recruit C-TAK1 to this compartment. Both Pim-1 and C-TAK1 underwent autophosphorylation, but only Pim-1 was able to phosphorylate C-TAK1 but not vice versa. Mass spectrometry analysis of C-TAK1 suggested that the sites of autophosphorylation and Pim-1-mediated phosphorylation are distinct and not overlapping. Phosphorylation by Pim-1 decreased C-TAK1 kinase activity significantly, in particular its ability to phosphorylate and inactivate Cdc25C, a protein that actively promotes cell cycle progression at the G(2)/M phase. Hence our findings directly suggest a novel role for Pim-1 as a positive regulator at the G(2)/M transition of the cell cycle.     

The two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (F) protein is a major contributor to this antigenic relatedness

The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (> or =6.0 log(10)) in the upper respiratory tract of hamsters and to moderate titers (> or =3.6 log(10)) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with postinfection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 expressing the fusion F protein of the CAN83 strain developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This result indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tracts of rhesus macaques but induced high levels of HMPV-neutralizing antibodies in serum effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tracts of African green monkeys, and the infected animals developed a high level of HMPV serum-neutralizing antibodies (1:500 to 1:1,000) effective against both lineages. Reciprocal cross-neutralization assays in which postinfection sera from all three primate species were used indicated that CAN75 and CAN83 are 64 to 99% related antigenically. HMPV-infected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results from hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross-neutralization in vitro.     

Recombinant human Metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate

Human metapneumovirus (HMPV) has recently been identified as a significant cause of serious respiratory tract disease in humans. In particular, the emerging information on the contribution of HMPV to pediatric respiratory tract disease suggests that it will be important to develop a vaccine against this virus for use in conjunction with those being developed for human respiratory syncytial virus and the human parainfluenza viruses. A recently described reverse genetic system (S. Biacchesi, M. H. Skiadopoulos, K. C. Tran, B. R. Murphy, P. L. Collins, and U. J. Buchholz, Virology 321:247-259, 2004) was used to generate recombinant HMPVs (rHMPVs) that lack the G gene, the SH gene, or both. The DeltaSH, DeltaG, and DeltaSH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Thus, the SH and G proteins are not essential for growth in cell culture. Apart from the absence of the deleted protein(s), the virions produced by the gene deletion mutants were similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. When administered intranasally to hamsters, the DeltaG and DeltaSH/G mutants replicated in both the upper and lower respiratory tracts, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were at least 40-fold and 600-fold restricted in replication in the lower and upper respiratory tract, respectively, compared to wild-type rHMPV. They also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Surprisingly, G is dispensable for protection, and the DeltaG and DeltaSH/G viruses represent promising vaccine candidates. In contrast, DeltaSH replicated somewhat more efficiently in hamster lungs compared to wild-type rHMPV (20-fold increase on day 5 postinfection). This indicates that SH is completely dispensable in vivo and that its deletion does not confer an attenuating effect, at least in this rodent model.     

Isomaltose formation by free and immobilized dextransucrase

The acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL-B512F with glucose as acceptor is of technical interest for isomaltooligosaccharide (IMOs) synthesis. Different experimental conditions were investigated for free and immobilized enzyme. The data for oligosaccharide formation up to a degree of polymerization 4 were correlated with a model developed earlier, and optimal reaction conditions for immobilized dextransucrase design and application were identified for later continuous application. Furthermore, stability was investigated for free and immobilized enzyme including stabilization by sugars.