Enzymatic synthesis of α-2-deoxyglucosyl derivatives catalyzed by organic solvent-resistant α-glucosidase

Organic solvent-resistant Aspergillus niger α-glucosidase (ANGase) can synthesize α-2-deoxyglucosyl derivatives (2DDs) in water-organic solvent media by a trans-addition reaction from d-glucal to various acceptors. Herein, we studied the influence of four different solvents on ANGase stability and activity. ANGase exhibited 47 or 43% residual activity following incubation in 50% (v/v) or in 70% (v/v) acetone for 4 h, respectively. When various carbohydrates were used as acceptor molecules, ANGase catalyzed the addition reaction of four different sugar alcohols, glucose, sucrose, or trehalose to d-glucal. Among the acceptor molecules tested, xylitol was the best acceptor by producing the highest yield (87% addition). The concentration of acetone/acceptor influenced the formation of 2DDs and the yields. We confirmed the molecular weight of five kinds of products by mass spectrometry and enzymatic hydrolysis. Current method is useful for the production of carbohydrates containing 2-deoxyglucose moiety.     

Presence and gradual disappearance of filaria-specific urinary IgG4 in babies born to antibody-positive mothers: a 2-year follow-up study

A total of 14 Sri Lankan pregnant women, who were anti-Brugia pahangi urinary IgG4 positive, and their 14 newborn babies were followed up for the urinary antibody for 2 years by enzyme-linked immunosorbent assay. Eight babies showed positive IgG4 reaction, at least once within 4 months after birth. Urinary antibody titers of mothers and their babies measured around the perinatal period showed a significant positive correlation, suggesting that baby's IgG4 was transferred from the mother through the placenta. The IgG4 decreased gradually and became negative in all positive babies by day 339.3 after birth. The present result provides a basis to judge if a positive urine ELISA test among babies is due to a new filarial infection.     

Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (r Si -HLP). r Si -HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1β and TNF-α) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of r Si -HLP was heat-unstable. In subsequent studies, r Si -HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that r Si -HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si -HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.     

Expanding GPCR homology model binding sites via a balloon potential: A molecular dynamics refinement approach

Homology modeling of G protein-coupled receptors is becoming a widely used tool in drug discovery. However, unrefined models built using the bovine rhodopsin crystal structure as the template, often have binding sites that are too small to accommodate known ligands. Here, we present a novel systematic method to refine model active sites based on a pressure-guided molecular dynamics simulation. A distinct advantage of this approach is the ability to introduce systematic perturbations in model backbone atoms in addition to side chain adjustments. The method is validated on two test cases: (1) docking of retinal into an MD-relaxed structure of opsin and (2) docking of known ligands into a homology model of the CCR2 receptor. In both cases, we show that the MD expansion algorithm makes it possible to dock the ligands in poses that agree with the crystal structure or mutagenesis data.     

Fruitless and doublesex coordinate to generate male-specific neurons that can initiate courtship

Biologists postulate that sexual dimorphism in the brain underlies gender differences in behavior, yet direct evidence for this has been sparse. We identified a male-specific, fruitless (fru)/doublesex (dsx)-coexpressing neuronal cluster, P1, in Drosophila. The artificial induction of a P1 clone in females effectively provokes male-typical behavior in such females even when the other parts of the brain are not masculinized. P1, located in the dorsal posterior brain near the mushroom body, is composed of 20 interneurons, each of which has a primary transversal neurite with extensive ramifications in the bilateral protocerebrum. P1 is fated to die in females through the action of a feminizing protein, DsxF. A masculinizing protein Fru is required in the male brain for correct positioning of the terminals of P1 neurites. Thus, the coordinated actions of two sex determination genes, dsx and fru, confer the unique ability to initiate male-typical sexual behavior on P1 neurons.     

Detection of anaerobic ammonium-oxidizing bacteria in Ago Bay sediments

We identified 16S rRNA gene sequences in sediment samples from Ago Bay in Japan, forming a new branch of the anammox group or closely related to anaerobic ammonium oxidizing (anammox) bacterial sequences. Anammox activity in the sediment samples was detected by (15)N tracer assays. These results, along with the results of fluorescence in situ hybridization (FISH) analysis, suggest the presence of anammox bacteria in the marine sediments.     

RNA-binding protein hoip accelerates polyQ-induced neurodegeneration in Drosophila

Abnormal polyglutamine (polyQ) expansion in the N-terminal domain of the human androgen receptor (hAR) is known to cause spinobulbar muscular atrophy (SBMA), a hereditary human neurodegenerative disorder. To explore the molecular mechanisms of neurodegeneration in SBMA, we genetically screened modulators of neurodegeneration in a Drosophila SBMA experimental model system. We identified hoip as an accelerator of polyQ-induced neurodegeneration. We found that hoip forms a complex with 18s rRNA together nop56 and nop5 proteins, whose human homologs are known to form a snoRNP complex involved in ribosomal RNA processing. Significantly, the levels of mutant polyQ-hAR were up-regulated in a mutant line overexpressing hoip. Consistently, severe neurodegeneration phenotype (rough eye) was also observed in both nop56 and nop5 overexpression mutant lines. These findings suggest that the process of neurodegeneration induced by abnormal polyQ expansion in the hAR may be regulated by the activity of snoRNP complex.     

Characterization of a dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri

The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD(+) as a specific electron acceptor, and the apparent K(m) values for DLA and NAD(+) were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K(m) values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 degrees C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 degrees C.