白蚁及其共生菌来源的4种木质纤维素酶的共表达

天然的木质纤维素材料含有纤维素、半纤维素和木质素等成分。降解天然木质纤维素底物时,需要木质纤维素酶共同作用。近年在木质纤维素酶的相互协同作用方面的研究引起人们的关注,成为一个新的研究热点,文中使用两个不同的共表达载体pETDuet-1和pRSFDuet-1,在大肠杆菌中共表达了白蚁及其肠道微生物来源的β-葡萄糖苷酶、内切β-1,4-葡聚糖酶、漆酶和木聚糖酶这4种木质纤维素酶,经过SDS-PAGE分析得到了与理论值一致的蛋白条带,同时经过酶活验证,这4种蛋白都具有酶活性。以磷酸处理的微晶纤维素(PASC)为底物,测定了共表达酶粗酶液与单独表达酶混合液的协同作用因子,从还原糖的产量上经计算共表达的粗酶液比单独表达酶的混合液对PASC的降解协同作用提高44%;以滤纸和磷酸处理的玉米芯为底物,测定降解协同作用,分别提高了34%和20%。结果表明,共表达酶的降解效率要高于混合的单组分酶液降解效率的总和。     

多级逆流工艺促进城市污泥厌氧发酵生产挥发性脂肪酸

采用一种新型的厌氧发酵工艺——多级逆流发酵工艺对城市污泥进行厌氧发酵, 实现高效产挥发性脂肪酸的目的。结果表明, 实验条件下应用多级逆流发酵工艺, 挥发性脂肪酸浓度与产率分别达到(10.5±0.5) g/L和0.20 gVFAs/ gVS, 与普通厌氧发酵工艺相比, 分别提高了31%和54%。此外, 在多级逆流工艺中, 底物有机质去除率可达50%, 较普通厌氧发酵提高了37%。进一步分析多级逆流工艺产酸的机制, 发现产酸效率的提高在于降低了发酵产物对厌氧产酸细菌的抑制效应, 并且工艺的VFAs产率以及有机质去除率分别取决于第一级和第三级厌氧发酵过程。因此, 城市污泥采用多级逆流工艺厌氧发酵不仅能够有效促进挥发性脂肪酸的生成, 而且能够较大程度上提高污泥中有机质的去除率。     

Effects of Bone Marrow-Derived Mesenchymal Stem Cells on the Axonal Outgrowth through Activation of PI3K/AKT Signaling in Primary Cortical Neurons Followed Oxygen-Glucose Deprivation Injury

Background

Transplantation with bone marrow-derived mesenchymal stem cells (BMSCs) improves the survival of neurons and axonal outgrowth after stroke remains undetermined. Here, we investigated whether PI3K/AKT signaling pathway is involved in these therapeutic effects of BMSCs.

Methodology/Principal Findings

(1) BMSCs and cortical neurons were derived from Sprague-Dawley rats. The injured neurons were induced by Oxygen–Glucose Deprivation (OGD), and then were respectively co-cultured for 48 hours with BMSCs at different densities (5×10³, 5×10⁵/ml) in transwell co-culture system. The average length of axon and expression of GAP-43 were examined to assess the effect of BMSCs on axonal outgrowth after the damage of neurons induced by OGD. (2) The injured neurons were cultured with a conditioned medium (CM) of BMSCs cultured for 24 hours in neurobasal medium. During the process, we further identified whether PI3K/AKT signaling pathway is involved through the adjunction of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a specific phosphatidylinositide-3-kinase (PI3K) inhibitor). Two hours later, the expression of pAKT (phosphorylated AKT) and AKT were analyzed by Western blotting. The length of axons, the expression of GAP-43 and the survival of neurons were measured at 48 hours.

Results

Both BMSCs and CM from BMSCs inreased the axonal length and GAP-43 expression in OGD-injured cortical neurons. There was no difference between the effects of BMSCs of 5×10⁵/ml and of 5×10³/ml on axonal outgrowth. Expression of pAKT enhanced significantly at 2 hours and the neuron survival increased at 48 hours after the injured neurons cultured with the CM, respectively. These effects of CM were prevented by inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions/Significance

BMSCs promote axonal outgrowth and the survival of neurons against the damage from OGD in vitro by the paracrine effects through PI3K/AKT signaling pathway.     

Effective approach to greatly enhancing selective secretion and expression of three cytoplasmic enzymes in Escherichia coli through synergistic effect of EDTA and lysozyme

An effective approach to greatly enhancing the selective secretion and expression of recombinant cytoplasmic enzymes in Escherichia coli was successfully developed through the synergistic effect of ethylenediaminetetraacetate (EDTA) and lysozyme. The method was applied to two endoglucanases (EGs) and an amylase. The optimal culture conditions of temperature and concentration of isopropyl-β-D: -1-thiogalactopyranoside (IPTG) were 23-30?°C and 0.2?mM, respectively, under which the three enzymes could be expressed in active form. Among all the chemicals tested, EDTA was found to be most suitable for enhancing the secretion of EG-I-1A into the medium. Addition of lysozyme alone had little influence on the secretion and expression. In contrast, on the basis of the addition of 5?g?EDTA/L at the induction time of 12?h, the simultaneous addition of 0.15?g?lysozyme/L further significantly increased the secretion and expression of the three enzymes, demonstrating the synergistic effect of EDTA and lysozyme. The production of EG-I-1A in the culture medium by adding 5?g?EDTA/L and 0.15?g?lysozyme/L under the optimal culture conditions of 23?°C and 0.2?mM IPTG was over 260-fold higher than that without EDTA and lysozyme under the standard conditions of 37?°C and 1?mM IPTG. In summary, the advantage of this novel cultivation approach for secretion was that not only did it selectively enhance the secretion of the proteins of interest, but also greatly increased the expression of the three enzymes by over 80?%.     

Exosomal miR-532-5p induced by long-term exercise rescues blood–brain barrier function in 5XFAD mice via downregulation of EPHA4

The breakdown of the blood–brain barrier, which develops early in Alzheimer's disease (AD), contributes to cognitive impairment. Exercise not only reduces the risk factors for AD but also confers direct protection against cognitive decline. However, the exact molecular mechanisms remain elusive, particularly whether exercise can liberate the function of the blood–brain barrier. Here, we demonstrate that long-term exercise promotes the clearance of brain amyloid-β by improving the function of the blood–brain barrier in 5XFAD mice. Significantly, treating primary brain pericytes or endothelial cells with exosomes isolated from the brain of exercised 5XFAD mice improves cell proliferation and upregulates PDGFRβ, ZO-1, and claudin-5. Moreover, exosomes isolated from exercised mice exhibit significant changes in miR-532-5p. Administration or transfection of miR-532-5p to sedentary mice or primary brain pericytes and endothelial cells reproduces the improvement of blood–brain barrier function. Exosomal miR-532-5p targets EPHA4, and accordingly, expression of EphA4 is decreased in exercised mice and miR-532-5p overexpressed mice. A specific siRNA targeting EPHA4 recapitulates the effects on blood–brain barrier-associated cells observed in exercised 5XFAD mice. Overall, our findings suggest that exosomes released by the brain contain a specific miRNA that is altered by exercise and has an impact on blood–brain barrier function in AD.     

Analysis of secondary structure and self‐assembly of amelogenin by variable temperature circular dichroism and isothermal titration calorimetry

Amelogenin is a proline‐rich enamel matrix protein known to play an important role in the oriented growth of enamel crystals. Amelogenin self‐assembles to form nanospheres and higher order structures mediated by hydrophobic interactions. This study aims to obtain a better insight into the relationship between primary–secondary structure and self‐assembly of amelogenin by applying computational and biophysical methods. Variable temperature circular dichroism studies indicated that under physiological pH recombinant full‐length porcine amelogenin contains unordered structures in equilibrium with polyproline type II (PPII) structure, the latter being more populated at lower temperatures. Increasing the concentration of rP172 resulted in the promotion of folding to an ordered β‐structured assembly. Isothermal titration calorimetry dilution studies revealed that at all temperatures, self‐assembly is entropically driven due to the hydrophobic effect and the molar heat of assembly (ΔH_A) decreases with temperature. Using a computational approach, a profile of domains in the amino acid sequence that have a high propensity to assemble and to have PPII structures has been identified. We conclude that the assembly properties of amelogenin are due to complementarity between the hydrophobic and PPII helix prone regions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of hypoxia-induced release of corticotropin-releasing factor in the rat hypothalamus by norepinephrine

Corticotropin-releasing factor (CRF) peptide release was activated by hypoxia in the rat hypothalamus. The mechanisms, however, of the hypoxia-induced CRF release remains unclear. In this study, we demonstrated that the norepinephrine (NE) and its receptors in the paraventricular nucleus (PVN) mediated the CRF release in a simulated altitude hypoxia. When rats were exposed to 5 or 7 km altitude of hypoxia for a short or long term: (1) NE levels in the PVN and the CeA, using the HPLC analysis, were intensity and time course dependently increased, but the increase in the PVN were potential than in the CeA. Restraint-induced NE increase was much higher in both the PVN and the CeA, compared with hypoxia-induced response. (2) Hypoxia and restraint significantly enhanced CRF release in the ME and the PVN but not in the CeA, through RIA assay, which result in stimulating corticosterone secretion. (3) Hypoxia-induced CRF release was reversed by an injection of prazosin (i.c.v.), an alpha-1 adrenoceptor antagonist, while administration of yohimbine (i.c.v.), an alpha-2 receptor antagonist, facilitated further CRF release. These data suggested that hypoxia induced NE activation centrally, via alpha-1 and -2 receptors, leading to improving hypothalamic CRF release, which in turn stimulated pituitary and adrenal cortex. Restraint presented much potential action on NE activation than hypoxia.     

Synthesis and anti-viral activity of a series of d- and l-2'-deoxy-2'-fluororibonucleosides in the subgenomic HCV replicon system

Based on the discovery of (2'R)-d-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of d- and l-2'-deoxy-2'-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2'-fluoro group, was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2'R)-d-2'-deoxy-2',5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhibited inhibition of ribosomal RNA. As N(4)-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N(4)-hydroxyl and the 2'-fluoro into one molecule, resulting (2'R)-d-2'-deoxy-2'-fluoro-N(4)-hydroxycytidine (23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.