Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.     

Decrease in the particle size of low-density lipoprotein (LDL) by oxidation

A radical reaction of low-density lipoprotein (LDL) causes fragmentation and cross-link of apolipoprotein B-100 (apoB). LDL (50 microg/ml) was subjected to the well-studied oxidation with Cu(2+) (1.67 microM). The concentration of alpha-tocopherol decreased to 10% of the initial level during the first 30 min. After this lag time, the conjugated diene content, as measured by absorption at 234 nm, started increasing and the residual apoB at 512 kDa determined by immunoblot after SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) was also decreased. The particle size of LDL determined by nondenaturing gradient gel electrophoresis decreased steadily during the initial 120 min, when residual native apoB was only 30% of the initial level. Plasma was also oxidized with Cu(2+) (400 microM). Under this condition, a clear lag time was not observed and alpha-tocopherol content, apoB, and the LDL particle size were decreased simultaneously. Based on these experiments, we propose that an oxidation reaction is involved in the formation of small dense LDL.     

Hyaluronan-binding motif identified by panning a random peptide display library

The glycosaminoglycan hyaluronan (HA) is involved in a variety of functions such as cell migration, adhesion, activation of intracellular signaling, metastasis, inflammation and wound repair. These functions of HA are mediated via HA-binding proteins (HABPs). To derive details of the HA-binding site in HABPs, here, we panned a random peptide display library expressed on the E. coli flagellin protein using HA-coated plates. Using this random peptide display library, 40 positive clones were obtained and the nucleotide sequences were determined. As a result, an Arg-Arg sequence, in addition to the known B-X7-B motif, was found to bind to HA. A binding experiment using the IAsys resonant mirror biosensor verified that a peptide containing an Arg-Arg sequence binds to HA.     

CD24 is expressed specifically in the nucleus pulposus of intervertebral discs

Intervertebral disc (IVD) consists of a soft gelatinous material in its center, the nucleus pulposus (NP), bounded peripherally by fibrocartilage, annulus fibrosus (AF). Despite the number of patients with IVD degeneration, gene expression analysis has not been undertaken in NP and therefore little is known about the molecular markers expressed in NP. Here, we undertook a microarray screen in NP with the other nine tissues to identify the specific cell surface markers for NP. Five membrane associating molecules out of 10,490 genes were identified as highly expressing genes in NP compared with the other tissues. Among them, we identified CD24, a glycosylphosphatidylinositol (GPI) anchor protein as a cell surface marker for NP. CD24 expression was also detected in the herniated NP and chordoma, a malignant primary tumor derived from notochordal cells, while it was absent in chondrosarcoma. Therefore, CD24 is a molecular marker for NP as well as the diseases of IVD.     

Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes

Abstract We constructed a mesophilic anaerobic chemostat that was continuously fed with synthetic wastewater containing propionate as the sole source of carbon and energy. Steady-state conditions were achieved below the critical dilution rate of 0.3 d ⁻¹ with almost complete substrate degradation. The propionate-degrading methanogenic communities in the chemostat at dilution rates of 0.01, 0.08, and 0.3 d ⁻¹ were analyzed using molecular biological techniques. Fluorescence in situ hybridization with archaeal and bacterial domain-specific probes showed that archaeal cells predominated throughout the three dilution rates. Archaeal-16S rRNA gene clone library analysis and quantitative real-time polymerase chain reaction studies showed that hydrogenotrophic methanogen rRNA genes closely related to Methanoculleus was detected at a dilution rate of 0.01 d ⁻¹ , whereas rRNA genes closely related to the Methanoculleus and Methanospirillum genera were detected at dilution rates of 0.08 and 0.3 d ⁻¹ . The aceticlastic methanogen, Methanosaeta , was detected throughout the three dilution rates. Bacterial-rRNA gene clone library analysis and denaturing gradient gel electrophoresis demonstrated that rRNA genes affiliated with the genus Syntrophobacter predominated at the low dilution rate, whereas rRNA genes affiliated with the phylum Firmicutes predominated at the higher dilution rates. A significant number of rRNA genes affiliated with the genus Pelotomaculum were detected at dilution rate of 0.3 d ⁻¹ . The diversity of genes encoding acetate kinase agreed closely with the results of the rRNA gene analysis. The dilution rates significantly altered the archaeal and bacterial communities in the propionate-fed chemostat.     

Follicular dendritic cells emerge from ubiquitous perivascular precursors

The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor β (PDGFRβ). PDGFRβ-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRβ(+)-derived cells abolished FDC, indicating that FDC originate from PDGFRβ(+) cells. Lymphotoxin-α-overexpressing prion protein (PrP)(+) kidneys developed PrP(+) FDC after transplantation into PrP(-) mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRβ(+) stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin β receptor (LTβR)(-) kidney capsules, differentiated into Mfge8(+)CD21/35(+)FcγRIIβ(+)PrP(+) FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRβ(+) FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation.     

Tiliroside, a glycosidic flavonoid, ameliorates obesity-induced metabolic disorders via activation of adiponectin signaling followed by enhancement of fatty acid oxidation in liver and skeletal muscle in obese-diabetic mice

Tiliroside contained in several dietary plants, such as rose hips, strawberry and raspberry, is a glycosidic flavonoid and possesses anti-inflammatory, antioxidant, anticarcinogenic and hepatoprotective activities. Recently, it has been reported that the administration of tiliroside significantly inhibited body weight gain and visceral fat accumulation in normal mice. In this study, we evaluated the effects of tiliroside on obesity-induced metabolic disorders in obese-diabetic KK-A(y) mice. In KK-A(y) mice, the administration of tiliroside (100 mg/kg body weight/day) for 21 days failed to suppress body weight gain and visceral fat accumulation. Although tiliroside did not affect oxygen consumption, respiratory exchange ratio was significantly decreased in mice treated with tiliroside. In the analysis of metabolic characteristics, it was shown that plasma insulin, free fatty acid and triglyceride levels were decreased, and plasma adiponectin levels were increased in mice administered tiliroside. The messenger RNA expression levels of hepatic adiponectin receptor (AdipoR)-1 and AdipoR2 and skeletal muscular AdipoR1 were up-regulated by tiliroside treatment. Furthermore, it was indicated that tiliroside treatment activated AMP-activated protein kinase in both the liver and skeletal muscle and peroxisome proliferator-activated receptor α in the liver. Finally, tiliroside inhibited obesity-induced hepatic and muscular triglyceride accumulation. These findings suggest that tiliroside enhances fatty acid oxidation via the enhancement adiponectin signaling associated with the activation of both AMP-activated protein kinase and peroxisome proliferator-activated receptor α and ameliorates obesity-induced metabolic disorders, such as hyperinsulinemia and hyperlipidemia, although it does not suppress body weight gain and visceral fat accumulation in obese-diabetic model mice.