Structure of the O-polysaccharide of Aeromonas hydrophila O:34; a case of random O-acetylation of 6-deoxy-L-talose

The O-polysaccharide of Aeromonas hydrophila O:34 was obtained by mild-acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy before and after O-deacetylation. The polysaccharide was found to contain D-Man, D-GalNAc and 6-deoxy-L-talose (L-6dTal), and the following structure of the tetrasaccharide repeating unit was established [carbohydrate structure see text] where 6dTal(I) is O-acetylated stoichiometrically at position-2 and 6dTal(II) carries no, one or two O-acetyl groups at any positions.     

Human milk oligosaccharides: an enzymatic protection step simplifies the synthesis of 3'- and 6'-O-sialyllactose and their analogues

We describe a chemo-enzymatic synthesis of 3'- and 6'-O-sialyllactose, two trisaccharides occurring in the 'acidic fraction' of the human milk oligosaccharides and endowed with potential antiadhesive activity. The key step is the highly regioselective 6'-O-acylation of benzyllactoside, which gave access to suitably protected lactose building blocks to be used as acceptors in the sialylation reaction. Moreover, the synthesis of the carboxymethyl and sulfo analogues of the title compounds is reported.     

Membrane-bound neuregulin1 type III actively promotes Schwann cell differentiation of multipotent Progenitor cells

Many steps of peripheral glia development appear to be regulated by neuregulin1 (NRG1) signaling but the exact roles of the different NRG1 isoforms in these processes remain to be determined. While glial growth factor 2 (GGF2), a NRG1 type II isoform, is able to induce a satellite glial fate in neural crest stem cells, targeted mutations in mice have revealed a prominent role of NRG1 type III isoforms in supporting survival of Schwann cells at early developmental stages. Here, we investigated the role of NRG1 isoforms in the differentiation of Schwann cells from neural crest-derived progenitor cells. In multipotent cells isolated from dorsal root ganglia, soluble NRG1 isoforms do not promote Schwann cell features, whereas signaling by membrane-associated NRG1 type III induces the expression of the Schwann cell markers Oct-6/SCIP and S100 in neighboring cells, independent of survival. Thus, axon-bound NRG1 might actively promote both Schwann cell survival and differentiation.     

Application of kinetic-based biospecific affinity chromatographic systems to ATP-dependent enzymes: studies with yeast hexokinase

This study is concerned with the development of kinetic-based bioaffinity chromatographic systems for purification of ATP-dependent kinases, with a particular focus on the allosteric yeast hexokinase enzyme (EC 2.7.1.1). Synthesis and characterization of highly substituted N(6)-linked and S(6)-linked immobilized ATP derivatives are described using a rapid solid-phase modular approach. Evaluation of the new immobilized ATP derivatives has been carried out using model chromatographic studies with yeast hexokinase, employing specific substrate analogues (N-acetyl-D-glucosamine and suramin) to promote biospecific adsorption, in the presence and absence of citrate (a so-called allosteric activator of hexokinase activity). In this paper, successful bioaffinity chromatography systems were developed for yeast hexokinase and, as a result, interesting binding and catalytic properties of the enzyme were highlighted and explored. The overall results confirm the potential for extrapolation of the kinetic locking-on tactic, a general kinetic-based bioaffinity approach already developed for the NAD(P)(+)-dependent dehydrogenases, to ATP/ADP-dependent enzymes. However, in view of the enhancement of the intrinsic ATPase activity of hexokinase with glucosamine derivatives, and the coincidental hydrolysis of immobilized ATP to immobilized ADP, future developments necessary to support adaptation of the approach to ATP-dependent enzymes are discussed.     

Rat liver glucose-6-phosphatase system: light scattering and chemical characterization

Glucose-6-phosphatase is a multicomponent system located in the endoplasmic reticulum, involving both a catalytic subunit (G6PC) and several substrate and product carriers. The glucose-6-phosphate carrier is called G6PT1. Using light scattering, we determined K(D) values for phosphate and glucose transport in rat liver microsomes (45 and 33mM, respectively), G6PT1 K(D) being too low to be estimated by this technique. We provide evidence that phosphate transport may be carried out by an allosteric multisubunit translocase or by two distinct proteins. Using chemical modifications by sulfhydryl reagents with different solubility properties, we conclude that in G6PT1, one thiol group important for activity is facing the cytosol and could be Cys(121) or Cys(362). Moreover, a different glucose-6-phosphate translocase, representing 20% of total glucose-6-phosphate transport and insensitive to N-ethylmaleimide modification, could coexist with liver G6PT1. In the G6PC protein, an accessible thiol group is facing the cytosol and, according to structural predictions, could be Cys(284).     

Analysis of genetic effects of major genes and polygenes on quantitative traits I. Genetic model for diploid plants and animals

A genetic model was proposed to simultaneously investigate genetic effects of both polygenes and several single genes for quantitative traits of diploid plants and animals. Mixed linear model approaches were employed for statistical analysis. Based on two mating designs, a full diallel cross and a modified diallel cross including F₂, Monte Carlo simulations were conducted to evaluate the unbiasedness and efficiency of the estimation of generalized least squares (GLS) and ordinary least squares (OLS) for fixed effects and of minimum norm quadratic unbiased estimation (MINQUE) and Henderson III for variance components. Estimates of MINQUE (1) were unbiased and efficient in both reduced and full genetic models. Henderson III could have a large bias when used to analyze the full genetic model. Simulation results also showed that GLS and OLS were good methods to estimate fixed effects in the genetic models. Data on Drosophila melanogaster from Gilbert were used as a worked example to demonstrate the parameter estimation. Received: 11 November 2000 / Accepted: 2 May 2001     

ε-Caprolactam-degradation by Alcaligenes faecalis for bioremediation of wastewater of a nylon-6 production plant

Alcaligenes faecalis G utilized 95–97% of 5–15 g -caprolactam l^–1 in 24–48 h over a pH range of 6–8.5 and at 23–40 °C, without complex nutrient requirement. In the absence of KH₂PO₄ and K₂HPO₄/MgSO₄ in the medium, only 7.6% and 0.2% of 10 g caprolactam l^–1 was utilized, respectively. The chemical oxygen demand (COD) of the wastewater of nylon-6 plant was mainly due to its caprolactam content. A. faecalis G decreased the caprolactam content and COD of the wastewater by 80–90% of the original in spite of the wastewater having higher caprolactam content (3600 mg l^–1) and COD (7700 mg l^–1) than those of any of the previous reports.     

Different genetic backgrounds influence the secretory expression of the LKA1-encoded Lipomyces kononenkoae α-amylase in industrial strains of Saccharomyces cerevisiae

A haploid laboratory strain and four industrial (baking, brewing, wine, ATCC) strains of Saccharomyces cerevisiae were transformed with the Lipomyces kononenkoae -amylase-encoding gene (LKA1). These transformants displayed significant differences in terms of the level of secretory expression of LKA1 under control of the PGK1 promoter and terminator, as well as their ability to produce and secrete the LKA1-encoded rawstarch-degrading -amylase and to ferment starch. These results demonstrate the importance of the selection of appropriate host strains for yeast development pursuant to starch conversion into commercially important commodities via consolidated bioprocessing.     

A site-specific mutation of tyrosine hydroxylase reduces feedback inhibition by dopamine in genetically modified cells grafted in parkinsonian rats

Aromatic L-amino acid decarboxylase (AADC) is necessary for conversion of L-DOPA to dopamine. Therefore, AADC gene therapy has been proposed to enhance pharmacological or gene therapies delivering L-DOPA. However, addition of AADC to the grafts of genetically modified cells expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1), which produce L-DOPA in parkinsonian rats, resulted in decreased production of L-DOPA and dopamine owing to feedback inhibition of TH by dopamine. End-product feedback inhibition has been shown to be mediated by the regulatory domain of TH, and site-specific mutation of serine 40 makes TH less susceptible to dopamine inhibition. Therefore, we investigated the efficacy of using TH with serine 40 mutated to leucine (mTH) in an ex vivo gene-therapy paradigm. Primary fibroblasts (PF) from Fischer 344 rats were transduced with retrovirus to express mTH or wild-type rat TH cDNA (wtTH). Both cell types were also transduced with GCH1 to provide the obligate TH cofactor, tetrahydrobiopterin. PF transfected with AADC were used as coculture and cografting partners. TH activities and L-DOPA production in culture were comparable between PFwtTHGC and PFmTHGC cells. In cocultures with PFAADC cells, PFmTHGC cells showed significant reduction in the inhibitory effect of dopamine compared with PFwtTHGC cells. In vivo microdialysis measurement showed that cografting PFAADC cells with PFmTHGC cells resulted in smaller decreases in L-DOPA and no reduction in dopamine levels compared with cografts of PFAADC cells with PFwtTHGC cells, which decreased both L-DOPA and dopamine levels. Maintenance of dopamine levels with lower levels of L-DOPA would result in more focused local delivery of dopamine and less potential side-effects arising from L-DOPA diffusion into other structures. These data support the hypothesis that mutation of serine 40 attenuates TH end-product inhibition in vivo and illustrates the importance of careful consideration of biochemical pathways and interactions between multiple genes in gene therapy.