Oleic acid decreases the expression of a cholesterol transport-related protein (NPC1L1) by the induction of endoplasmic reticulum stress in CaCo-2 cells

The reported data indicate that oleic acid (OA) decreases cholesterol absorption. To explore the underlying mechanisms, the effects of OA on the expression of cholesterol transport-related proteins (NPC1L1, ABCG5/8, ACAT2, MTP) and the unfolded protein response (UPR) pathway were studied in CaCo-2 enterocytes by incubating CaCo-2 cells with taurocholate micelles or taurocholate micelles containing different concentrations of OA (0.25–1.0 mM). We show that OA effectively induces XBP1 mRNA splicing, a key component of the UPR signaling, and the expression of BiP and mature ATF6 proteins in a concentration-dependent manner, leading to the induction of endoplasmic reticulum (ER) stress and activation of the UPR. Interestingly, OA decreases NPC1L1 expression in a dose-dependent manner while it has no effects on ABCG5 and MTP mRNA level or SREBP-2, ABCG8, and ACAT2 protein level. In CaCo-2 cells treated with 1.0 mM OA, both the NPC1L1 mRNA level and the NPC1L1 protein expression in brush-border membrane fractions were decreased by 39% and 37%, respectively (P < 0.01). A dose of 1 mM dithiothreitol (DTT), a positive control for ER stress induction, also decreases NPC1L1 mRNA and protein expression by 27% and 23%, respectively (P < 0.05). Furthermore, 4-phenyl-butyric acid, an UPR inhibitor, blocks OA- and DTT-induced reduction on NPC1L1 mRNA and protein levels. The results suggest that OA down-regulates NPC1L1 mRNA and protein expression via the induction of the UPR, which may play an important role in reducing intestinal cholesterol absorption.     

Overexpression of small glutamine-rich TPR-containing protein promotes apoptosis in 7721 cells

It is known that small glutamine-rich TPR-containing protein (SGT) is the member of TPR motif family. However, the biological functions of SGT remain unclear. In this paper, we report that SGT plays a role in apoptotic signaling. Ectopic expression of SGT enhances DNA fragment and nucleus breakage after the induction of apoptosis. Increasing mRNA level of SGT is also observed in 7721 cells undergoing apoptosis, knockdown the expression of endogenous SGT contributes to the decrease of apoptosis of 7721 cells. Deletion analysis reveals that TPR domain is critical to pro-apoptotic function of SGT. Furthermore, we demonstrated that the PARP cleavage and cytochrome c release are enhanced when SGT is overexpressed in 7721 cells during apoptosis. Collectively, our results indicate that SGT is a new pro-apoptotic factor.     

Effects of pH and calcium ions on the conformational transitions in silk fibroin using 2D Raman correlation spectroscopy and 13C solid-state NMR

Silk fibroin exists in a number of different states, such as silk I and silk II, with different properties largely defined by differences in secondary structure composition. Numerous attempts have been made to control the transitions from silk I to silk II in vitro to produce high-performance materials. Of all the factors influencing the structural compositions, pH and some metal ions play important roles. This paper focuses on the influence of pH and Ca(2+) ions on the conformational transition from silk I to silk II in regenerated (redissolved) Bombyx mori fibroin. One- and two-dimensional correlation Raman spectroscopy was used to describe qualitatively the transitions in secondary structure in silk I, silk II, and their intermediates as pH and Ca(2+) ion concentration were changed, while (13)C cross polarization magic angle spinning (CP/MAS) solid-state NMR was used to quantify these changes. We showed that conditions (low pH, pH 5.2; a defined range of Ca(2+) ion concentrations; gradual water removal) that mimic natural silk spinning promote the formations of beta-sheet and distorted beta-sheet characteristic of silk II or silk II-related intermediate. In contrast, higher pH (pH 6.9-8.0) and higher Ca(2+) ion concentrations maintain "random coil" conformations typical of silk I or silk I-related intermediate. These results help to explain why the natural silk spinning process is attended by a reduction in pH from 6.9 to 4.8 and a change in the Ca(2+) ion concentration in the gland lumen as fibroin passes from the posterior division through the secretory pathway to the anterior division.     

Down-regulation of p21WAF1 promotes apoptosis in senescent human fibroblasts: involvement of retinoblastoma protein phosphorylation and delay of cellular aging

It has been suggested that genes which exercise checkpoint control during cell cycle traverse are equally important to the process of apoptotic cell death. In this study, we show that the key cell cycle regulatory gene p21(WAF1) is also involved in the execution of apoptosis. p21(WAF1) expression was down-regulated during NaBu-induced apoptosis of senescent normal diploid human 2BS fibroblasts. Conversely, when p21(WAF1) expression was actively suppressed in 2BS cells by a stably transfected antisense p21(WAF1) construct, apoptosis was accelerated and senescence was delayed, as shown by several markers of cell aging. Down-regulation of p21(WAF1) by antisense caused an increase in the phosphorylation and inactivation of pRb. Phosphorylation of pRb was further enhanced upon induction of apoptosis by NaBu. Our results suggest that p21(WAF1), acting through the phosphorylation of pRb, regulates whether 2BS cells cease to proliferate and become senescent but resistant to apoptosis, or whether they accelerate proliferation while becoming more susceptible to apoptotic stimuli.     

GST/ AEP 融合蛋白原核表达载体的构建、表达及鉴定

目的：为进一步研究抗癫痫肽（And—epilepsy peptide,AEP）的抗痫机制及筛选其相关作用蛋白,进行GST／AEP融合蛋白原核表达载体的构建及融合蛋白的表达。方法：通过PCR基因扩增对AEP基因进行扩增,并将其克隆于谷胱甘肽-S-转移酶（GST）融合蛋白表达质粒pGEX-4T-1中,经酶切、序列鉴定分析后,用该重组质粒转化大肠杆菌B121（DE3）,经IPTG诱导获得表达,并采用Western Blot进行检测。结果：成功构建了AEP原核表达载体,并在大肠杆菌B121中获得表达。结论：成功构建了GST／AEP原核表达载体,并表达了GST／AEP融合蛋白。     

A new species of Leptocycas(Zamiaceae)from the Upper Triassic sediments of Liaoning Province,China

A new cycad,Leptocycas yangcaogouensis sp.nov.,was found in sediments from the Late Triassic in western Liaoning,China.The pinnately compound leaves(Pseudoctenis type)are screwed in a crown on the stem top.The leaflets are linear,with parallel veins and decurrent bases on the rachis.The leaf bases are persistent.The cataphylls intermix with the leaves.The female cone is ovoid in shape.The characteristics of the new plant are more similar to those of Leptocycas gracilis,a Triassic cycad from North America,but the new species differs from L.gracilis in the size of its stem(7-8 vs.3-5 cm in diameter,respectively),leaves(length × width 100 × 16 vs.30 × 7 cm,respectively)and leaf density along the stem(4-6 vs.1-2 bases/1 cm length,respectively).Both L.gracilis and L.yangcaogouensis,having leaves of the Pseudoctenis type,show a closer relationship to the extant Dioon of Zamiaceae.The present study provides evidence for the origin of the genus Dioon,which may have come from Leptocycas plants of the Triassic.It would be assumed that the extent cycads in Zamiaceae originate from the pteridosperms in the Late Paleozoic and have evolved through the stage of L.gracilis and L.yangcaogouensis in Late Triassic,and reaching the extant Dioon.     

Convenient synthesis of sulfated oligofucosides

Two types of sulfated octyl tetra- to octaoligofucosides with different sulfation patterns were synthesized employing a combination of stepwise elongation and convergent strategies in which trichloroacetimidates and thioglycosides were selected as the glycosyl donors.     

RACK1 recruits STAT3 specifically to insulin and insulin-like growth factor 1 receptors for activation, which is important for regulating anchorage-independent growth

Current understanding of the activation of STATs is through binding between the SH2 domain of STATs and phosphotyrosine of tyrosine kinases. Here we demonstrate a novel role of RACK1 as an adaptor for insulin and insulin-like growth factor 1 receptor (IGF-1R)-mediated STAT3 activation specifically. Intracellular association of RACK1 via its N-terminal WD domains 1 to 4 (WD1-4) with insulin receptor (IR)/IGF-1R is augmented upon respective ligand stimulation, whereas association with STAT3 is constitutive. Purified RACK1 or RACK1 WD1-4 associates directly with purified IR, IGF-1R, and STAT3 in vitro. Insulin induces multiprotein complex formation of RACK1, IR, and STAT3. Overexpression or downregulation of RACK1 greatly enhances or decreases, respectively, IR/IGF-1R-mediated activation of STAT3 and its target gene expression. Site-specific mutants of IR and IGF-1R impaired in RACK1 binding are ineffective in mediating recruitment and activation of STAT3 as well as in insulin- or IGF-1-induced protection of cells from anoikis. RACK1-mediated STAT3 activation is important for insulin and IGF-1-induced anchorage-independent growth in certain ovarian cancer cells. We conclude that RACK1 mediates recruitment of STAT3 to IR and IGF-1R specifically for activation, suggesting a general paradigm for the need of an adaptor in mediating activation of STATs by receptor protein tyrosine kinases.