Synthesis and antifungal activity of 1H-indole-4,7-diones

1H-Indole-4,7-diones were synthesized and tested for in vitro antifungal activity against fungi. The synthesized 1H-indole-4,7-diones generally showed good antifungal activity against Candida krusei, Cryptococcus neoformans, and Aspergillus niger. The results suggest that 1H-indole-4,7-diones would be potent antifungal agents.     

Effects of daunorubicin on ganglioside expression and neuronal differentiation of mouse embryonic stem cells

Gangliosides are implicated in neuronal development processes. The regulation of ganglioside levels is closely related to the induction of neuronal cell differentiation. In this study, the relationship between ganglioside expression and neuronal cell development was investigated using an in vitro model of neural differentiation from mouse embryonic stem (mES) cells. Daunorubicin (DNR) was applied to induce the expression of gangliosides in embryoid body (EB) (4+). We observed an increase in expression of gangliosides in all stages of EBs by treatment of DNR (2microM). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GD3, GD1a, GT1b, and GQ1b increased in DNR-treated 7-day-old EB (4+) [EB (4+):7]. DNR treatment significantly increased the expression of gangliosides, especially GT1b and GQ1b in comparison to control cells. Interestingly, GQ1b co-localized with microtubule-associated protein 2 (MAP-2) expressing cells in DNR-treated EB (4+):7. The co-localization of GQ1b and MAP-2 was found in neurite-bearing cells in DNR-treated 15-day-old EB (4+) [EB (4+):15], whereas no significant expression of GQ1b and less neurite formation were observed in untreated control. Also, the expression of synaptophysin and NF200, both neuronal markers associated with neruites, was increased by DNR treatment. These results demonstrate that DNR increases expression of gangliosides, especially GQ1b, in differentiating neuronal cells. Further, neurite-bearing neuronal cell differentiation can be facilitated by DNR, possibly through the induction of gangliosides. Thus, the present data suggest that DNR is beneficial for facilitating neuronal differentiation from ES cells and among the gangliosides analyzed in the present study, GQ1b is mainly involved in neurite formation.     

Biological and biochemical modulation of Trichomonas vaginalis KT9 isolate after shifting of culture medium from TPS-1 into TYM

To evaluate the biological and biochemical characteristics of Trichomonas vaginalis KT9 isolate, the growth and size of trichomonads, pathogenicity in mouse, protein profiles and proteinase activity were examined after shifting the medium from TPS-1 into TYM. Generation time of trichomonads in TYM medium was 4.5 hr in comparison to TPS-1 with 7.1 hr. Size of trichomonads cultured in TPS-1 medium (8.5 ± 0.9 × 6.0 ± 0.9 µm) was significantly smaller than those in TYM medium (10.9 ± 1.4 × 8.2 ± 0.9 µm). Trichomonads cultured in TYM medium produced subcutaneous abscess in 9 out of 10 mice, whereas those in TPS-1 medium produced abscesses in 2 out of 10 mice. In SDS-PAGE, trichomonad lysates from both media showed ten common bands. However, trichomonads in TYM medium showed additional bands of 136 kDa, 116 kDa and 40 kDa in comparison to those in TPS-1 with 100 kDa. By immunoblot with T. vaginalis-immunized rabbit sera, T. vaginalis cultivated in both TYM and TPS-1 media showed 5 common bands, and unique bands of 116 kDa, 105 kDa, and 86 kDa were observed in trichomonads in TYM while a 140 kDa band in those in TPS-1. In gelatin SDS-PAGE, trichomonads in TYM degraded gelatin stronger than those in TPS-1. Also protease activity of trichomonads in TYM was significantly higher than that of trichomonads in TPS-1 using Bz-Pro-Phe-Arg-Nan as a substrate. According to the results, it is assumed that the shift from TPS-1 into TYM medium for cultivation of T. vaginalis might modulate the biological and biochemical properties of T. vaginalis in vitro.     

How do organic solvents affect peroxidase structure and function?

The effect of organic solvents on horseradish peroxidase structure and function has been studied. Some, but not complete, enzyme denaturation occurs even in low volumes of water-miscible organic solvents (e.g., greater than 30% v/v dioxane, greater than 50% v/v methanol, and greater than 20% v/v acetonitrile) as determined by the decreased difference between the fluorescence of peroxidase's sole tryptophan residue and free L-tryptophan in solution. Absorbance and electron paramagnetic resonance spectroscopies indicate exposure of peroxidase's active site to the organic solvent. This reduces the local polarity in the enzyme's active site and results in stronger hydrogen bonding of phenolic substrates to the enzyme. In extreme cases (e.g., 95% v/v dioxane, 90% v/v acetonitrile, and ethyl and butyl acetate containing 2 and 1% v/v aqueous buffer, respectively), the transition state of the enzymic reaction is sufficiently perturbed so as to alter the magnitude of the Hammett rho value. This is most likely the result of the increased strength of hydrogen bonding between electron-donating alkoxyphenols (negative sigma values) and an electrophilic group in the enzyme's active site, thereby reducing catalytic efficiencies for such substrates relative to alkyl- and chlorophenols. Perhaps the most important effect of the organic solvent, however, is the significant ground-state stabilization of phenolic substrates in organic media as opposed to aqueous buffer. This stabilization can account for nearly 4 orders of magnitude in reduction of catalytic efficiency and is manifested in increased Km's. This study indicates that enzymes can maintain much of their native active-site structure in organic media and that the effect of solvent on substrate thermodynamics must be considered.     

Purification and characterization of a cytosolic, 42-kDa and Ca2+-dependent phospholipase A2 from bovine red blood cells: its involvement in Ca2+-dependent release of arachidonic acid from mammalian red blood cells

It has become evident that a Ca(2+)-dependent release of arachidonic acid (AA) and subsequent formation of bioactive lipid mediators such as prostaglandins and leukotrienes in red blood cells (RBCs) can modify physiological functions of neighboring RBCs and platelets. Here we identified a novel type of cytosolic PLA(2) in bovine and human RBCs and purified it to apparent homogeneity with a 14,000-fold purification. The purified enzyme, termed rPLA(2), has a molecular mass of 42 kDa and reveals biochemical properties similar to group IV cPLA(2), but shows different profiles from cPLA(2) in several column chromatographies. Moreover, rPLA(2) did not react with any of anti-cPLA(2) and anti-sPLA(2) antibodies and was identified as an unknown protein in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Divalent metal ions tested exhibited similar effects between rPLA(2) and cPLA(2), whereas mercurials inhibited cPLA(2) but had no effect on rPLA(2). Antibody against the 42-kDa protein not only precipitated the rPLA(2) activity, but also reacted with the 42-kDa protein from bovine and human RBCs in immunoblot analysis. The 42-kDa protein band was selectively detected in murine fetal liver cells known as a type of progenitor cells of RBCs. It was found that EA4, a derivative of quinone newly developed as an inhibitor for rPLA(2), inhibited a Ca(2+) ionophore-induced AA release from human and bovine RBCs, indicating that this enzyme is responsible for the Ca(2+)-dependent AA release from mammalian RBCs. Finally, erythroid progenitor cell assay utilizing diaminobenzidine staining of hemoglobinized fetal liver cells showed that rPLA(2) detectable in erythroid cells was down-regulated when differentiated to non-erythroid cells. Together, our results suggest that the 42-kDa rPLA(2) identified as a novel form of Ca(2+)-dependent PLA(2) may play an important role in hemostasis, thrombosis, and/or erythropoiesis through the Ca(2+)-dependent release of AA.     

The role of AiiA, a quorum-quenching enzyme from Bacillus thuringiensis, on the rhizosphere competence

Bacteria sense their population density and coordinate the expression of target genes, including virulence factors in Gram-negative bacteria, by the N-acylhomoserine lactones (AHLs)-dependent quorum-sensing (QS) mechanism. In contrast, several soil bacteria are able to interfere with QS by enzymatic degradation of AHLs, referred to as quorum quenching. A potent AHL-degrading enzyme, AiiA, of Bacillus thuringiensis has been reported to effectively attenuate the virulence of bacteria by quorum quenching. However, little is known about the role of AiiA in B. thuringiensis itself. In the present study, an aiiA-defective mutant was generated to investigate the role of AiiA in rhizosphere competence in the root system of pepper. The aiiA mutant showed no detectable AHL-degrading activity and was less effective for suppression of soft-rot symptom caused by Erwinia carotovora on the potato slice. On the pepper root, the survival rate of the aiiA mutant significantly decreased over time compared with that of wild type. Interestingly, viable cell count analysis revealed that the bacterial number and composition of E. carotovora were not different between treatments of wild type and the aiiA mutant, although root application of the aiiA mutant in pepper failed to protect the plant from root rot. These results provide evidence that AiiA can play an important role in rhizosphere competentce of B. thuringiensis and bacterial quorum quenching to Gram-negative bacteria without changing bacterial number or composition.     

Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label‐free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT‐PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor‐β (TGF‐β) signaling changed during adipogenesis. The expressions of secreted frizzled‐related proteins, dickkopf‐related proteins, and latent TGF‐β‐binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF‐β signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity‐related metabolic diseases.     

An antioxidant system required for host protection against gut infection in Drosophila

A fundamental question that applies to all organisms is how barrier epithelia efficiently manage continuous contact with microorganisms. Here, we show that in Drosophila an extracellular immune-regulated catalase (IRC) mediates a key host defense system that is needed during host-microbe interaction in the gastrointestinal tract. Strikingly, adult flies with severely reduced IRC expression show high mortality rates even after simple ingestion of microbe-contaminated foods. However, despite the central role that the NF-kappaB pathway plays in eliciting antimicrobial responses, NF-kappaB pathway mutant flies are totally resistant to such infections. These results imply that homeostasis of redox balance by IRC is one of the most critical factors affecting host survival during continuous host-microbe interaction in the gastrointestinal tract.     

Interaction of profilin‐1 and F‐actin via a β‐arrestin‐1/JNK signaling pathway involved in prostaglandin E2‐induced human mesenchymal stem cells migration and proliferation

Although many previous reports have examined the function of prostaglandin E₂ (PGE₂) in the migration and proliferation of various cell types, the role of the actin cytoskeleton in human mesenchymal stem cells (hMSCs) migration and proliferation has not been reported. The present study examined the involvement of profilin‐1 (Pfn‐1) and filamentous‐actin (F‐actin) in PGE₂‐induced hMSC migration and proliferation and its related signal pathways. PGE₂ (10^?6 M) increased both cell migration and proliferation, and also increased E‐type prostaglandin receptor 2 (EP2) mRNA expression, β‐arrestin‐1 phosphorylation, and c‐Jun N‐terminal kinase (JNK) phosphorylation. Small interfering RNA (siRNA)‐mediated knockdown of β‐arrestin‐1 and JNK (‐1, ‐2, ‐3) inhibited PGE₂‐induced growth of hMSCs. PGE₂ also activated Pfn‐1, which was blocked by JNK siRNA, and induced F‐actin level and organization. Downregulation of Pfn‐1 by siRNA decreased the level and organization of F‐actin. In addition, specific siRNA for TRIO and F‐actin‐binding protein (TRIOBP) reduced the PGE₂‐induced increase in hMSC migration and proliferation. Together, these experimental data demonstrate that PGE₂ partially stimulates hMSCs migration and proliferation by interaction of Pfn‐1 and F‐actin via EP2 receptor‐dependent β‐arrestin‐1/JNK signaling pathways. J. Cell. Physiol. 226: 559–571, 2011. © 2010 Wiley‐Liss, Inc.     

Chromosome aberrations in workers of nuclear-power plants

The frequencies of chromosome aberrations in 135 workers from nuclear-power plants were compared with those in 135 age-matched controls. A total of 135,000 cells was scored. The frequencies of dicentric chromosome were 1.67 × 10⁻³ in the exposed group and 0.49 × 10⁻³ in the control group and those of chromosome-type deletion were 3.33 × 10⁻³ and 1.10 × 10⁻³, respectively. The frequencies of all types of chromosome aberrations in the exposed subjects were higher than those in the control group, but no significant trend of dose-dependent increase was observed when only the exposed group were considered. Poisson regression analysis, with both exposed and control included, showed that there was a significant association of chromosome aberration with radiation dose and the duration of work, but not with age, smoking habit and alcohol intake. It was also found that recent exposure to radiation, within the last 5 years, had contributed more to the observed chromosome aberration than earlier exposure.