Polymerization of cardanol using soybean peroxidase and its potential application as anti-biofilm coating material

Soybean peroxidase (20 mg) catalyzed the oxidative polymerization of cardanol in 2-propanol/phospate buffer solution (25 ml, 1:1 v/v) and yielded 62% polycardanol over 6 h. Cobalt naphthenate (0.5% w/w) catalyzed the crosslinking of polycardanol and the final hardness of crosslinked polycardanol film exceeded 9 H scale as pencil scratch hardness, which shows a high potential as a commercial coating material. In addition, it showed an excellent anti-biofouling activity to Pseudomonas fluorescens compared to other polymeric materials such as polypropylene.     

An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius

Genes homologous to 2-deoxystreptamine (DOS) biosynthetic genes were isolated from aminoglycoside producers, Micromonospora and Streptomyces spp., using PCR primers based on the core sequences of 2-deoxy-scyllo-inosose (DOI) synthase and L-glutamine: scyllo-inosose aminotransferase (GIA). Identities of 40-45% were observed for DOI synthases, and 65-75% were observed for GIAs. The gene cluster of tobramycin biosynthesis was isolated from the genomic library of Streptomyces tenebrarius using DOI synthase as a probe. Sequencing of 33.9 kb revealed 24 putative open reading frames including the tobramycin biosynthetic gene cluster (13.8 kb) and a transport protein. This cluster encodes proteins homologous to 2-deoxystreptamine biosynthetic enzymes, glycosyltransferase and other aminocyclitols biosynthetic enzymes. Sequence analysis revealed the evolution of DOI synthases from 3-dehydroquinate (DHQ) synthases in actinomycetes. DOI synthases and GIA are therefore useful for cloning biosynthetic genes of DOS-containing aminocyclitol antibiotics or for screening such metabolites producers.     

Plant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts

Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l^–1 myo-inositol, 4.4 M BA, and 1.4 M 2,4-D) at a density of 5×10⁴ protoplasts ml^–1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l^–1 myo-inositol was replaced with the same osmolarity of 90 g l^–1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l^–1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Tolerance is dependent on complement C3 fragment iC3b binding to antigen-presenting cells

Systemic tolerance can be induced by the introduction of antigen into an immune-privileged site. Here we investigated the role of complement in the induction of tolerance after intraocular injection. We found that the development of antigen-specific tolerance is dependent on a complement activation product. The ligation of the complement C3 activation product iC3b to complement receptor type 3 (the iC3b receptor) on antigen-presenting cells resulted in the sequential production of transforming growth factor-beta2 and interleukin-10, which is essential for the induction of tolerance. These observations may extend to the development of both neonatal tolerance and other forms of acquired tolerance.     

Role of Reactive Oxygen Species and Glutathione in Inorganic Mercury-Induced Injury in Human Glioma Cells

The present study was undertaken to examine the role of reactive oxygen species (ROS) and glutathione (GSH) in glia cells using human glioma cell line A172 cells. HgCl₂ caused the loss of cell viability in a dose-dependent manner. HgCl₂-induced loss of cell viability was not affected by H₂O₂ scavengers catalase and pyruvate, a superoxide scavenger superoxide dismutase, a peroxynitrite scavenger uric acid, and an inhibitor of nitric oxide N^G-nitro-arginine Methyl ester. HgCl₂ did not cause changes in DCF fluorescence, an H₂O₂-sensitive fluorescent dye. The loss of cell viability was significantly prevented by the hydroxyl radical scavengers dimethylthiourea and thiourea, but it was not affected by antioxidants DPPD and Trlox. HgCl₂-induced loss of cell viability was accompanied by a significant reduction in GSH content. The GSH depletion was almost completely prevented by thiols dithiothreitol and GSH, whereas the loss of viability was partially prevented by these agents. Incubation of cells with 0.2 mM buthionine sulfoximine for 24 hr, a selective inhibitor of -glutamylcysteine synthetase, resulted in 56% reduction in GSH content without any change in cell viability. HgCl² resulted in 34% reduction in GSH content, which was accompanied by 59% loss of cell viability. These results suggest that HgCl₂-induced cell death is not associated with generation of H₂O₂ and ROS-induced lipid peroxidation. In addition, these data suggest that the depletion of endogenous GSH itself may not play a critical role in the HgCl₂-induced cytotoxicity in human glioma cells.     

Identification of a novel human member of the DEAD box protein family

The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM). From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family. DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division. RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family. Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets. In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 μm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5±2.8% with a linear range of 0.1–100 μg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

In vivo Tracking of Dendritic Cell using MRI Reporter Gene,Ferritin

The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R₂*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R₂*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R₂* in both in vivo and ex vivo T₂*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines.     

Molecular Evidence for an Old World Origin of Galapagos and Caribbean Band-Winged Grasshoppers (Acrididae: Oedipodinae: Sphingonotus)

Patterns of colonization and diversification on islands provide valuable insights into evolutionary processes. Due to their unique geographic position and well known history, the Galapagos Islands are an important model system for evolutionary studies. Here we investigate the evolutionary history of a winged grasshopper genus to infer its origin and pattern of colonization in the Galapagos archipelago. The grasshopper genus Sphingonotus has radiated extensively in the Palaearctic and many species are endemic to islands. In the New World, the genus is largely replaced by the genus Trimerotropis. Oddly, in the Caribbean and on the Galapagos archipelago, two species of Sphingonotus are found, which has led to the suggestion that these might be the result of anthropogenic translocations from Europe. Here, we test this hypothesis using mitochondrial and nuclear DNA sequences from a broad sample of Sphingonotini and Trimerotropini species from the Old World and New World. The genetic data show two distinct genetic clusters representing the New World Trimerotropini and the Old World Sphingonotini. However, the Sphingonotus species from Galapagos and the Caribbean split basally within the Old World Sphingonotini lineage. The Galapagos and Caribbean species appear to be related to Old World taxa, but are not the result of recent anthropogenic translocations as revealed by divergence time estimates. Distinct genetic lineages occur on the four investigated Galapagos Islands, with deep splits among them compared to their relatives from the Palaearctic. A scenario of a past wider distribution of Sphingonotus in the New World with subsequent extinction on the mainland and replacement by Trimerotropis might explain the disjunct distribution.     

Hyponatremia Predicts New-Onset Cardiovascular Events in Peritoneal Dialysis Patients

Background and Aim

Cardiovascular (CV) disease is the leading cause of morbidity and mortality in patients on peritoneal dialysis (PD). Hyponatremia was recently shown to be a modifiable factor that is strongly associated with increased mortality in PD patients. However, the clinical impact of hyponatremia on CV outcomes in these patients is unclear.

Methods

To determine whether a low serum sodium level predicts the development of CV disease, we carried out a prospective observational study of 441 incident patients who started PD between January 2000 and December 2005. Time-averaged serum sodium (TA-Na) levels were determined to investigate the ability of hyponatremia to predict newly developed CV events in these patients.

Results

During a mean follow-up of 43.2 months, 106 (24.0%) patients developed new CV events. The cumulative incidence of new-onset CV events after the initiation of PD was significantly higher in patients with TA-Na levels ≤ 138 mEq/L than in those with a TA-Na > 138 mEq/L. After adjustment for multiple potentially confounding covariates, an increase in TA-Na level was found to be associated with a significantly lower risk of CV events (subdistribution hazard ratio per 1 mEq/L increase, 0.90; 95% confidence interval, 0.83–0.96; p = 0.003). Patients with a TA-Na ≤ 138 mEq/L had a 2.31-fold higher risk of suffering a CV event.

Conclusions

These results provide evidence of a clear association between low serum sodium and new-onset CV events after dialysis initiation in PD patients. Whether the correction of hyponatremia for this indication provides additional protection for the development of CV disease in these patients remains to be addressed in interventional studies.