Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-β-d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.     

AIB1 is required for the acquisition of epithelial growth factor receptor-mediated tamoxifen resistance in breast cancer cells

Acquired resistance to tamoxifen has become a serious obstacle in breast cancer treatment. The underlying mechanism responsible for this condition has not been completely elucidated. In this study, a tamoxifen-resistant (Tam-R) MCF-7 breast cancer cell line was developed to mimic the occurrence of acquired tamoxifen resistance as seen in clinical practice. Increased expression levels of HER1, HER2 and the estrogen receptor (ER)-AIB1 complex were found in tamoxifen-resistant cells. EGF stimulation and gefitinib inhibition experiments further demonstrated that HER1/HER2 signaling and AIB1 were involved in the proliferation of cells that had acquired Tam resistance. However, when AIB1 was silenced with AIB1-siRNA in Tam-R cells, the cell growth stimulated by the HER1/HER2 signaling pathway was significantly reduced, and the cells were again found to be inhibited by tamoxifen. These results suggest that the AIB1 protein could be a limiting factor in the HER1/HER2-mediated hormone-independent growth of Tam-R cells. Thus, AIB1 may be a new therapeutic target, and the removal of AIB1 may decrease the crosstalk between ER and the HER1/HER2 pathway, resulting in the restoration of tamoxifen sensitivity in tamoxifen-resistant cells.     

Functional reconstitution of the human serotonin receptor 5-HT6 using synthetic transmembrane peptides

Seven transmembrane (7TM) synthetic peptides mimicking the α-helical TM domains of the human serotonin receptor subtype-6 (5-HT₆) were autonomously reconstituted in detergent micelle and liposome environments. The degree of assembly of the 7TM peptides was characterized by monitoring the fluorescence resonance energy transfer (FRET) between donor and acceptor probes labeled at the amino termini of the second and fourth TM-peptides, respectively. The FRET efficiency of these peptides significantly increased when the 7TM peptides were reconstituted in liposome compare to detergent micelles. Furthermore, the 7TM peptides reconstituted in liposomes selectively bound to free serotonin and serotonin-conjugated magnetic beads, yielding a dissociation constant of 0.84 μM. These results show that the seven individual TM domains of 5-HT₆ can spontaneously assemble into liposomes in a conformation that mimics a native structure, and further demonstrate that specific interactions between TM helices play a critical role in the folding and stabilizing of GPCRs. The autonomous assembly of 7TM-peptides can be applied to the screening of agonists for GPCRs that are difficult to manipulate.     

A Phylogroup of the Siberian Chipmunk from Korea (Tamias sibiricus barberi) Revealed from the Mitochondrial DNA Cytochrome b Gene

In the full sequences of the mtDNA cytochrome b gene, 26 haplotypes (Tamias sibiricus barberi) from six localities of central and southern Korea were distinct from 21 haplotypes (Tamias sibiricus orientalis) from five localities of northeast China and Vladivostok, Russia. The average Tamura–Nei nucleotide distance between the subspecies (11.40%) and maximum infrasubspecific distances (3.74% and 4.72%) support the subspecies classification of T. s. barberi based on morphometric comparison. The 26 haplotypes of T. s. barberi were also distinct from 2 haplotypes of T. s. orientalis and Tamias sibiricus jacutensis from far-east Russia (average distance, 11.86%). Thus T. s. barberi constitutes a “phylogroup” (average nucleotide distance > 10%); analyses with nuclear genes of northeast Asian specimens, including North Korean ones, are necessary to clarify its taxonomic status. Furthermore, 49 haplotypes of T. sibiricus from eastern Asia differed from 19 haplotypes of another 18 Tamias spp. from America (weighted-average distance, 18.58%). T. sibiricus is, therefore, distinct enough to be recognized as a subgenus, Eutamias.     

Centrosome attachment to the C. elegans male pronucleus is dependent on the surface area of the nuclear envelope

A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein.     

The Semiquinone-Iron Complex of Photosystem II: Structural Insights from ESR and Theoretical Simulation; Evidence that the Native Ligand to the Non-Heme Iron Is Carbonate

The semiquinone-iron complex of photosystem II was studied using electron spin resonance (ESR) spectroscopy and density functional theory calculations. Two forms of the signal were investigated: 1), the native g ∼ 1.9 form; and 2), the g ∼ 1.84 form, which is well known in purple bacterial reaction centers and occurs in photosystem II when treated with formate. The g ∼ 1.9 form shows low- and high-field edges at g ∼ 3.5 and g < 0.8, respectively, and resembles the g ∼ 1.84 form in terms of shape and width. Both types of ESR signal were simulated using the theoretical approach used previously for the BRC complex, a spin Hamiltonian formalism in which the semiquinone radical magnetically interacts (J ∼ 1 cm⁻¹) with the nearby high-spin Fe²⁺. The two forms of ESR signal differ mainly by an axis rotation of the exchange coupling tensor (J) relative to the zero-field tensor (D) and a small increase in the zero-field parameter D (∼6 cm⁻¹). Density functional theory calculations were conducted on model semiquinone-iron systems to identify the physical nature of these changes. The replacement of formate (or glutamate in the bacterial reaction centers) by bicarbonate did not result in changes in the coupling environment. However, when carbonate (CO₃²⁻) was used instead of bicarbonate, the exchange and zero-field tensors did show changes that matched those obtained from the spectral simulations. This indicates that 1), the doubly charged carbonate ion is responsible for the g ∼ 1.9 form of the semiquinone-iron signal; and 2), carbonate, rather than bicarbonate, is the ligand to the iron.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.