Ethanol extract of fermented soybean, Chungkookjang, inhibits the apoptosis of mouse spleen, and thymus cells

Apoptosis is a step of the cell cycle which is important in the regulation of immune cell populations. Chungkookjang is a Korean traditional fermented soybean containing microorganisms, enzymes, and bioactive compounds which was used in the treatment of mouse spleen as well as thymus cells (CH1-fermented soybean containing barley, wormwood, and sea tangle; CH2-fermented soybean) and was found to exhibit substantially reduced small DNA fragmentation. An MTT assay showed that the treatment of CH1 and CH2 into the mouse splenocytes and thymocytes sharply increased their survival. Moreover, a FACS analysis also showed that CH1 and CH2 are effective at suppressing the apoptosis of splenocytes and thymocytes. The fermented soybean isoflavone concentrations, which are implicated in lowering breast and prostate cancers, lowering the risk of cardiovascular diseases, and improving bone health, were determined using Capillary Electrophoresis-Electrochemical Detection (CE-ED). The amount of Daidzein in fermented soybean significantly increased by 44-fold dramatically, compared with those in unfermented soybean. In this study, we demonstrated that ethanol extracts of Chungkookjang promote the survival of the mouse spleen and thymus cells in culture by suppressing their apoptotic death. Future studies should investigate which genes are related to apoptosis of the immune cells.     

Comparative proteomic analysis for hCTLA4Ig production in transgenic rice suspension cultures using two-dimensional difference gel electrophoresis

The new technology, two-dimensional difference gel electrophoresis (2D DIGE), uses fluorescent dyes to simplify the process of detecting and matching proteins between multiple gels by allowing for the separation of up to three separate protein samples within the same gel. In this study, recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4lg) was produced in transgenic rice suspension cell cultures and the intracellular proteins were analyzed by 2D DIGE. The highest level of hCTLA4Ig (25.4 mg/L) was obtained five days after induction. The intracellular proteins expressed at both the growth and induction culture stages were separated and analyzed using DeCyder software. At least 2,218 spots were detected with two-fold thresholds and 95% confidence. We found that 29 spots increased and 20 spots decreased in their intensities during the production of recombinant hCTLA4Ig. In addition, the 2D Western blot of hCTLA4Ig revealed that this fusion protein was expressed in a variety of isoforms.     

Identification and functional analysis of vibrio vulnificus SmcR, a novel global regulator

Recently, quorum sensing has been implicated as an important global regulator controlling the production of numerous virulence factors such as capsular polysaccharides in bacterial pathogens. The nucleotide and deduced amino acid sequences of smcR, a homolog of V. harveyi luxR identified from V. vulnificus ATCC29307, were analyzed. The amino acid sequence of SmcR from V. vulnificus was 72 to 92% similar to those of LuxR homologs from Vibrio spp. Functions of SmcR were assessed by the construction of an isogenic mutant, whose smcR gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of smcR resulted in a significant alteration in biofilm formation, in type of colony morphology, and in motility. When compared with the wild-type, the smcR mutant exhibited reduced survival under adverse conditions, such as acidic pH and hyperosmotic stress. The smcR mutant exhibited decreased cytotoxic activity toward INT 407 cells in vitro. Furthermore, the intraperitoneal LD50 of the smcR mutant was approximately 10(2) times higher than that of parental wild-type. Therefore, it appears that SmcR is a novel global regulator, controlling numerous genes contributing to the pathogenesis as well as survival of V. vulnificus.     

Ameliorating effect of Gardenia jasminoides extract on amyloid beta peptide-induced neuronal cell deficit

The brains of Alzheimer's disease (AD) patients are characterized by large deposits of amyloid beta peptide (Abeta). Abeta is known to increase free radical production in nerve cells, leading to cell death that is characterized by lipid peroxidation, free radical formation, protein oxi-dation, and DNA/RNA oxidation. In this study, we selected an extract of Gardenia jasminoides by screening, and investigated its ameliorating effects on Abeta-induced oxidative stress using PC12 cells. The effects of the extract were evaluated using the 2,7 -dichlorofluorescein diacetate (DCF-DA) assay and the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. To find the active component, the ethanol extract was partitioned with hexane, chloroform, and ethyl acetate, respectively, and the active component was purified by silica-gel column chromatography and HPLC. The results suggested that Gardenia jasminoides extract can reduce the cytotoxicity of Abeta in PC 12 cells, possibly by reducing oxidative stress.     

Cloning and functional expression of the gene encoding an inhibitor against <Emphasis Type="Italic">Aspergillus flavus</Emphasis> α-amylase,a novel seed lectin from <Emphasis Type="Italic">Lablab purpureus</Emphasis> (<Emphasis Type="Italic">Dolichos lablab</Emphasis>)

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.     

Molecular modeling study on the enantioselective binding of S- and R-ofloxacin to various DNA sequences

The complex formation of S- and R-ofloxacin with the self-complementary oligonucleotides, namely d[ATAGCGCTAT](2), d[GCGATATCGC](2) and d[ATAICICTAT](2), were investigated by the molecular dynamics (MD) simulation. Four starting positions, including two intercalation positions with different insertion directions and two minor groove binding positions, were considered. The total energy of both S- and R-ofloxacin-d[ATAGCGCTAT](2) complex, in which ofloxacin binds in the minor groove of the oligonucleotide, were lower than any intercalation binding mode. For both enantiomers, formation of the complex with GC oligonucleotide is more favorable than AT and IC oligonucleotides. When S- and R-ofloxacin are compared, the S-enantiomer exhibits more favorable total energy and torsion angles in the complex formation. This result is in agreement with the experimental observation [Hwangbo et al., Eur J Pharm Sci 18, 197 (2003)]. In the complex, both enantiomers form two hydrogen bonds: one between the carbonyl group of ofloxacin and the amine group of G16 and the other between the fluorine group and the G6 amine for S-ofloxacin. However, only one hydrogen bond is formed between endocyclic hydrogen atom at the C2 position of adenine and inosine base and carbonyl group of ofloxacin, which may be the reason for the GC preferentiality of ofloxacin.     

The suppressive effect of beta-glucan on the production of tumor necrosis factor-alpha in BV2 microglial cells

Beta-glucan was recently shown to have the ability to enhance and stimulate the immune system in humans, but little is known about its the anti-inflammatory effects. We investigated the effect of beta-glucan on the production of tumor necrosis factor-alpha (TNF-alpha), a major pro-inflammatory mediator, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. beta-Glucan decreased the production and expression of TNF-alpha. In addition, it blocked LPS-stimulated activation of nuclear factor kappa B (NF-kappaB). Hence beta-glucan might suppress LPS-stimulated TNF-alpha production by inhibiting NF-kappaB in BV2 microglial cells.     

Egg yolk soluble protein stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells

We determined the effects of yolk water-soluble protein (YSP) on bone formation in pre-osteoblastic MC3T3-E1 cells. YSP (50-5,000 microg/ml) increased cell proliferation and collagen content. Alkaline phosphatase (ALP) activity was also increased by YSP treatment. After enhancement of ALP activity, significant augmentation of calcification was observed. These results suggest that YSP is a promising agent for the prevention and treatment of bone loss.     

Protective effect of anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) against myelotoxicity induced by 5-fluorouracil

The toxicities associated with 5-fluorouracil (5-FU), a potent broad-spectrum chemotherapeutic agent, can not only affect the morbidity and the efficacy of chemotherapy but also limit its clinical use. The objective of this study is to investigate the effects of a commercial anthocyanin-rich extract from bilberry (AREB) against 5-FU-induced myelotoxicity in vivo, and against chemosensitivity to 5-FU in vitro. A single injection of 5-FU at 200 mg/kg induced severe peripheral erythrocytopenia, thrombocytopenia and leucopenia as well as hypocellularity of the spleen and bone marrow in C57BL/6 mice. Oral administration of 500 mg/kg of AREB for 10 days significantly increased the number of red blood cells, neutrophils, and monocytes in peripheral blood to 1.2-fold, 9-fold, and 6-fold, respectively, compared with those seen after treatment with 5-FU alone (p< 0.05-0.001). The hypocellularity of the spleen and bone marrow caused by 5-FU was also distinctly alleviated in the AREB-treated group. Furthermore, AREB treatment with 50 and 100 microg/ml as a monomeric anthocyanin did not interfere with, but rather enhanced the chemotherapeutic efficacy of 5-FU in vitro. These results suggest that AREB may have protective potential against 5-FU-induced myelotoxiciy and/or the ability to enhance the chemotherapeutic effectiveness of 5-FU.