Inactivation of TNF signaling by rationally designed dominant-negative TNF variants

Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.     

Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst

Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.     

Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase

Silent information regulator 2 (Sir2) proteins (sirtuins) are nicotinamide adenine dinucleotide-dependent deacetylases that regulate important biological processes. Mammals have seven sirtuins, Sirt1 to Sirt7. Four of them (Sirt4 to Sirt7) have no detectable or very weak deacetylase activity. We found that Sirt5 is an efficient protein lysine desuccinylase and demalonylase in vitro. The preference for succinyl and malonyl groups was explained by the presence of an arginine residue (Arg(105)) and tyrosine residue (Tyr(102)) in the acyl pocket of Sirt5. Several mammalian proteins were identified with mass spectrometry to have succinyl or malonyl lysine modifications. Deletion of Sirt5 in mice appeared to increase the level of succinylation on carbamoyl phosphate synthase 1, which is a known target of Sirt5. Thus, protein lysine succinylation may represent a posttranslational modification that can be reversed by Sirt5 in vivo.     

Effect of microfluidization on in vitro micellization and intestinal cell uptake of lutein from Chlorella vulgaris

Chlorella is a nutrient-rich microalga that contains protein, lipid, minerals, vitamins, and high levels of lutein. This study evaluated the bioavailability of lutein from Chlorella vulgaris using a coupled in vitro digestion and human intestinal Caco-2 cell model. Lutein bioaccessibility was low, and approximately 75% of total C. vulgaris lutein was not micellized during the digestion process but remained in the insoluble digestate. Microfluidization improved lutein micellization efficiency during C. vulgaris digestion. C. vulgaris was microfluidized at a pressure exceeding 10000 psi, and the cell surface disruption was visualized by scanning electron microscopy. The mean C. vulgaris particle size was reduced from 3.56 to 0.35 μm with the microfluidization treatment. C. vulgaris microfluidization at 20000 psi was three times more efficient for aqueous lutein micelles production as compared with untreated C. vulgaris, and the final lutein content accumulated by intestinal Caco-2 cells was also higher with microfluidization. C. vulgaris lutein stability was not affected by microfluidization. These results indicate that microfluidization may be useful for improving lutein bioaccessibility from C. vulgaris during food processing.     

Identification and structure elucidation of a p-phenoxybenzaldehyde in bamboo shoots by HPLC-ESI/MS/MS and NMR

In this study, a derivative of p-phenoxybenzaldehyde in bamboo shoots was investigated. Bamboo shoots were ground and extracted with water, and an aqueous suspension was purified by SPE using Oasis HLB cartridges. After the SPE procedure, the analytes were analyzed by HPLC with refractive index detection (HPLC-RI). In the HPLC-RI analysis for sucralose, a putative sucralose was detected. In the subsequent HPLC-PDA analysis, the suspicious peak showed a unique UV absorption spectrum with the maximum wavelength at 285 nm indicating the existence of an aromatic ring. The contents of the unknown compound in bamboo shoot products ranged from 0.01 to 0.15 mg/g. The identity of the unknown compound was further confirmed by HPLC-ESI/MS/MS. The molecular weight of the unknown compound was determined to be 244. The chemical structure of the unknown compound was elucidated on the basis of NMR spectroscopic analyses ((1)H, (13)C, DEPT, COSY, HMQC, and HMBC). Finally, the structure of the unknown compound was characterized as 4-(4-dihydroxymethylphenoxy)benzaldehyde.     

Occurrence of Honeysuckle Yellow Vein Virus (HYVV) containing a monopartite DNA-A genome in Korea

Two newly emerged begomoviruses were isolated from naturally infected tomato (Solanum lycopersicum) plants grown in greenhouses at Jeju Island and Dangjin in Korea and their genomes were characterized. These viruses-infected plants had very small leaves that curled upward, yellow margins and a leathery appearance, and a bushy and stunted appearance with short internodes. Nucleotide (nt) sequence analysis of their genomes showed that they have a DNA-A component of a monopartite begomovirus. Their genomes comprised 2763 and 2764 nucleotides with six open reading frames. The results of nt sequence similarity analysis of DNA-A genome between the two Korean isolates and isolates of Tobacco leaf curl Japan virus (TbLCJV), Honeysuckle yellow vein virus (HYVV), Honeysuckle yellow vein mosaic virus (HYVMV), and Eupatorium yellow vein virus in Japan (EpYVV) showed that they are likely similar to HYVV-[Masuda] (89.4–92.8% nt identity). Consequently, we tentatively propose the two isolates’ names as HYVV-Jeju and -DJ according to the ICTV geminivirus rules. Phylogenetic relationship analysis of 33 DNA-A genome sequences using PAUP* 4.0b10 and MrBayes revealed that HYVV-Jeju and -DJ belong to the Far East Asian begomovirus species complex. Within the Far East Asian begomovirus species complex, HYVV-Jeju and -DJ are distantly related to EpYVV, HYVMV, and TbLCJV groups. Based on the presence of a recombination fragment spanning the C3 ORF, a recombinant origin was suggested for both HYVV-Jeju and –DJ, with parents close to Japanese isolates HYVMV-[SP1:00] and Eupatorium yellow vein virus (EpYVV)-[Suya]. In addition, the presence of a further recombination fragment spanning the IR suggested the parents of HYVV-DJ were close to HYVV-Jeju and EpYVV-[Suya].     

Sweet and sour cherry phenolics and their protective effects on neuronal cells

The identification of phenolics from various cultivars of fresh sweet and sour cherries and their protective effects on neuronal cells were comparatively evaluated in this study. Phenolics in cherries of four sweet and four sour cultivars were extracted and analyzed for total phenolics, total anthocyanins, and their antineurodegenerative activities. Total phenolics in sweet and sour cherries per 100 g ranged from 92.1 to 146.8 and from 146.1 to 312.4 mg gallic acid equivalents, respectively. Total anthocyanins of sweet and sour cherries ranged from 30.2 to 76.6 and from 49.1 to 109.2 mg cyanidin 3-glucoside equivalents, respectively. High-performance liquid chromatography (HPLC) analysis revealed that anthocyanins such as cyanidin and peonidin derivatives were prevalent phenolics. Hydroxycinnamic acids consisted of neochlorogenic acid, chlorogenic acid, and p-coumaric acid derivatives. Glycosides of quercetin, kaempferol, and isorhamnetin were also found. Generally, sour cherries had higher concentrations of total phenolics than sweet cherries, due to a higher concentration of anthocyanins and hydroxycinnamic acids. A positive linear correlation (r2 = 0.985) was revealed between the total anthocyanins measured by summation of individual peaks from HPLC analysis and the total anthocyanins measured by the pH differential method, indicating that there was in a close agreement with two quantifying methods for measuring anthocyanin contents. Cherry phenolics protected neuronal cells (PC 12) from cell-damaging oxidative stress in a dose-dependent manner mainly due to anthocyanins. Overall results showed that cherries are rich in phenolics, especially in anthocyanins, with a strong antineurodegenerative activity and that they can serve as a good source of biofunctional phytochemicals in our diet.     

Antioxidative, antimutagenic, and anticarcinogenic activities of rice bran extracts in chemical and cell assays

Ethanol-water (70:30 v/v) extracts from rice brans removed from seeds of two blackish-purple pigmented (Sanhaehyanghyulla and Suwon 415) and one nonpigmented (Chuchung) brown rice cultivars were evaluated for antioxidative, anti-tumor-promoting, and anticarcinogenic activities in chemical assays and in mammalian cells (human leukemia HL-60, marmoset B lymphoblastoid B95-8, and Chinese hamster V79 lung cells) by the following tests: inhibition of xanthine oxidase activity; chelation of ferrous ions; reduction of potassium ferricyanide; scavenging of superoxide anions, hydroxyl radicals, and intracellular peroxides; inhibition of 4-nitroquinoline N-oxide-induced mutagenesis; and inhibition of phorbol ester-induced tumor promotion. The extracts from the pigmented rice seeds had generally higher activities in all tests than did the extract from the nonpigmented variety. The results suggest that brans from pigmented rice varieties may provide a source of new natural antioxidants and anticarcinogens and that such rice cultivars with high antioxidative potential also provide a genetic resource for the development of new, improved rice cultivars that may make it possible to enhance both the nutritional and medical value of rice-based diets.     

Polyozellin isolated from Polyozellus multiplex induces phase 2 enzymes in mouse hepatoma cells and differentiation in human myeloid leukaemic cell lines

Induction of cellular phase 2 detoxifying enzymes is associated with cancer preventive potential. Quinone reductase (QR) has been used as a prototype for anticarcinogenic phase 2 enzymes because of its widespread distribution in mammalian systems, large amplitude of inducer response, and ease of measurement in murine hepatoma cells. Methanol extract of Polyozellus multiplex, which shows a strong QR induction potential, was subjected to bioassay-guided fractionation, and polyozellin (PM1) appeared to be a major active component. In in vitro cultured cells (hepa1c1c7 and BPRc1 cells), polyozellin enhanced QR, glutathione S-transferase (GST) activities, and glutathione (GSH) content in a dose-dependent manner. The compound also significantly promoted differentiation of HL-60 human promyelocytic emia cells. In conclusion, polyozellin deserves further in vivo study to evaluate its potential as a cancer preventive agent.